JP2023522903A - Methods and compositions - Google Patents

Abstract

A method of stimulating muscle regeneration comprising delivering a CCR5 interacting agent or encoding molecule to muscle. CCR5 interacting agents bind to muscle stem cells and stimulate myoblast proliferation and muscle regeneration. One example of a CCR5 interacting agent is NAMPT or a derivative thereof containing a cytokine finger motif. Methods and compositions include cell compositions expressing CCR5 interacting agents, including satellite cells or macrophage cells or populations of progenitor/progenitor cells thereof, and their application in stem cell therapy, or muscle deficits, Includes use in treating disorders or injuries. The examples demonstrate muscle tissue regeneration with minimal fibrosis. A NAMPT polypeptide fragment that includes the C-terminal portion of NAMPT that includes the cytokine finger is also operable. The composition further comprises a NAMPT polypeptide fragment and one or more of tissue stem cells or macrophages or progenitor/progenitor cells, scaffolding or retention materials, tissue delivery-enhancing or cell retention moieties.

Description

The present disclosure relates to the technical field of productive tissue repair and regeneration, and treatment of subjects in need thereof.

Any reference in this specification to any prior document or to any known matter is an admission that such prior document or known matter forms part of the general general knowledge of the art to which this specification pertains. or acknowledgment or suggestion of any kind and should not be perceived as such.

Bibliographic details of the references referred to are listed at the end of the specification.

Skeletal muscle typically accounts for approximately 40% of human body weight. It is formed during growth by myogenesis, in which paired blocks of paraxial mesoderm known as somites become myogenic, which expand to form integrated, complex muscle tissue. Form. Growth, regeneration and repair of muscle tissue are driven by mesodermal-derived skeletal muscle resident stem cells throughout life.

Skeletal muscle typically accounts for approximately 40% of the body weight of an adult human. It is formed during growth by myogenesis, in which paired blocks of paraxial mesoderm known as somites become transient myoblasts that form muscle stem cells, which expand to form myotubes. It is integrated through the fusion of myoblasts to the surface to form a complex muscle tissue. During further stages of myogenesis, muscle stem cells (called satellite cells) migrate to occupy niches between the sarcoma and the basal layer of individual muscle fibers. Amniotes are born with a full set of muscle fibers, and as adults, muscle repair is generally accomplished by increasing the size of existing fibers. Throughout life, muscle tissue homeostasis, growth, regeneration and repair are driven by skeletal muscle resident stem cells of mesodermal origin. At the molecular level, quiescent satellite cells require and express the transcription factor PAX7 and also express PAX3. After skeletal muscle injury, some satellite cells are activated and proliferate to form myoblasts, which differentiate and fuse to form new muscle fibers or to form damaged muscle fibers. merge and heal. This myogenic program is controlled by the myogenic regulators MYF5, MYOD, MYOG and MRF4. Many other elements and cells associated with the muscle niche are also believed to be involved in the complex cellular processes and eventual production of functional tissue in the context of homeostasis and regeneration. Therefore, it has been difficult to identify the origin and nature of the signals that stimulate satellite cell activation and proliferation.

Satellite cells are prototypical in that they are unipotent, tissue-resident stem cells that occupy specific anatomical niches within differentiated tissues. Recent decades of research have revealed the unique ability of this system to effectively coordinate muscle repair in response to a wide variety of injuries. Despite this demonstrated regenerative capacity, transplantation of isolated muscle stem cells has not yet had therapeutic impact, and regenerative therapies that stimulate muscle stem cells are still lacking.

The present invention specifically provides compositions for use in providing myoblast-based therapies, thereby addressing a significant and diverse unmet clinical need.

The term "and/or", e.g., "X and/or Y", shall be understood to mean "X and Y" or "X or Y" for both meanings or for either should be understood to provide explicit support for the meaning of

As used herein, the term "about" refers to ±10%, or ±5% of the specified value, unless otherwise specified.

Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" are meant to include the recited element, integer or step, or group of elements, integers or steps, other elements , integers or steps, or groups of elements, integers or steps.

As used herein, the singular forms "a," "an," and "the" shall include singular and plural references unless the context dictates otherwise.

The present disclosure is based, in part, on the results of experiments using time-lapse photography to describe the temporal and cellular framework of muscle regeneration at the single-cell level. This is the first time that these interactions and pathways have been globally visualized in a vertebrate system, and the newly observed cellular behaviors and methods and compositions described herein may also be useful in other tissue types. What is reproducible is proposed and realized herein.

Certain macrophage subsets have been shown to play a surprisingly direct and important role in regulating tissue regeneration in vivo, i.e., a fraction of wound-attracted macrophages It was found that it forms a transient stem cell niche together with endogenous tissue stem cells and induces its activation. Ablation of this niche-specific subset of macrophages markedly reduces the number of proliferative progenitors present at the site of injury, resulting in regeneration defects. The term injury as used herein refers to replacing tissue lost or rebuilding/regenerating functional tissue due to any process, such as a disease process, the result of infection or trauma, longevity, poor diet or lack of exercise. It broadly refers to any wound, externally or internally inflicted or present, that requires tissue regeneration for the purpose.

An obligate satellite cell-macrophage niche within the wound was identified in real time to facilitate efficient skeletal muscle regeneration and injury repair. This indicates that some of the wound-attracted macrophages form a transient stem cell niche and are myogenic. Referring to FIG. 2m, it can be seen that macrophage cells with large fringes adhere to small satellite cells for a long period of time and divide repeatedly. Ablation of this niche-specific macrophage subset markedly reduces the number of myogenic precursors present at the site of injury, resulting in muscle regeneration defects.

In one embodiment, the stem cells are tissue stem cells, such as muscle stem cells. In one embodiment, the stem cells are skeletal muscle stem cells (satellite cells). Other muscle stem cells include cardiac tissue stem cells or non-muscle cells. As provided herein, in one embodiment the tissue stem cells express the CCR5 receptor.

Thus, along with their well-known ability to modulate pro- and anti-inflammatory events, specific macrophage populations also provide a transient stem cell activation niche (cells are spatially constrained together in muscle tissue and directly interact). Macrophages or macrophage-derived factors detailed herein are therefore proposed for their use in modulating stem cell activity, directly activating quiescent tissue stem cells, and in tissue regeneration.

One macrophage-derived factor described herein is nicotinamide phosphoribosyltransferase (also known as NAMPT, visfatin and PBEF (pre-B cell enhancing factor)). NAMPT is shown herein to be upregulated and produced by injury-resident host macrophages. Specific derivatives of NAMPT have been developed that interact with muscle stem cell receptors. A composition containing it was found to induce muscle stem cell proliferation and muscle regeneration (see Figures and Examples). In one embodiment, activation is through chemokine receptor binding. Other drugs that act specifically through this receptor also stimulate muscle regeneration (eg for CCR5, CCL8 and CCL4). Importantly, muscle treatment with NAMPT was associated with little or minimal fibrosis in a clinically relevant volumetric wound model. NAMPT and its functional derivatives are therefore proposed for their use to stimulate healing and improve the quality of healing in order to promote complete restoration of tissue function ie productive tissue repair and regeneration. . Reference herein to NAMPT refers to a full-length molecule (e.g., SEQ ID NO: 4), a functionally active portion or fragment thereof that activates resting tissue stem cells (functionally arranged or operably linked to multiple fragments), and derivatives thereof containing modifications suitable for production and clinical or commercial use known in the art, such as enhanced tissue delivery or enhanced signaling function. As described herein, the term also includes fusion proteins or conjugates comprising all or part of NAMPT that activate satellite cells. NAMPT includes orthologs and isoforms.

References to "functional derivatives" of NAMPT or NAMPTcif include full-length molecules and portions, fragments (including orthologs and homologues), monomers, dimers, trimers, tetramers or multimers of full-length NAMPT or NAMPTcif. Forms of peptides, and variants and other modified forms, functional derivatives that bind at least resting tissue stem cells and induce their signaling, activation and proliferation, as further described herein. . The present application provides new uses for known molecules, such as NAMPT, and also functional derivatives of known molecules for use in the compositions, methods or uses disclosed herein. The present invention provides novel molecules containing

In one aspect, the application provides compositions that provide chemokine receptor interaction or binding activity, or muscle tissue stem cell interaction activity for use in stimulating muscle regeneration. In one embodiment, the present application provides chemokine receptor interaction or binding activity or satellite cell binding or interaction activity for use in stimulating muscle regeneration without or substantially without fibrosis. Provided is a composition for

In one embodiment, the chemokine receptor is a tissue stem cell receptor that binds NAMPT, including the CCR5 chemokine receptor, or a tissue stem cell receptor that binds NAMPTcif. In one embodiment, compositions comprising cells or other agents that provide CCR5-interacting activity bind tissue stem cells, particularly muscle stem cells. Cells or agents may optionally be modified to enhance the selectivity of binding to particular tissue stem cells, as described herein, if desired. Cells or agents may optionally be modified to enhance cytokine receptor signaling as described herein.

The term "regeneration" in relation to muscle is used herein in a broad sense and refers to the flow-on effects on muscle and muscle-related tissue as a direct result of activation of muscle stem cells (also called satellite cells). include. Regeneration, therefore, includes muscle wound repair, muscle maintenance, growth, repair, and enhancement of the ability of muscle cells to multiply productively and form functional tissue. The term includes the generation of muscle tissue, and the repair of damaged muscle, the process of muscle regeneration (myogenesis) beginning with the activation and proliferation of muscle stem cells, proliferation of myoblasts, early differentiation into muscle cells and muscle growth. Associated with terminal differentiation into fibers. In one embodiment, regeneration is associated with minimal fibrosis that allows establishment of a native structure or regenerated tissue with normal or near-normal biological properties, rather than fibrotic or weakened tissue. Muscle function properties may be determined by standard tests of contractile muscle function, including tests for strength (eg, eccentric muscle contraction), power and endurance, and physical length and volume. The term also includes the growth of muscle tissue in industrial cultures.

In one non-limiting embodiment, enhancement of muscle stem cell chemokine receptor signaling is associated with muscle damage, including volumetric muscle loss injury or muscle degeneration/atrophy, or muscle or neuromuscular injury, muscle or neuromuscular degeneration. It is particularly useful in treating subjects having conditions, myopathies, or predisposition thereto. In one embodiment, the chemokine receptor binding activity is expressed in cells such as macrophages or stem cells that express chemokine receptor interactors/binding factors, as well as polypeptides or peptides, nucleic acid molecules, antibodies that have or encode chemokine receptor binding activity. or in the form of molecules such as receptor-interacting portions thereof, small molecules or other agents.

In one embodiment, the present application provides a method of stimulating stem cell proliferation, such as satellite cell proliferation. In one embodiment, the method comprises administering to the cell or subject an effective amount of a cell composition comprising macrophages having chemokine receptor agonist activity. In one embodiment, chemokine receptor agonist activity in one or more muscle stem cells stimulates or enhances muscle stem cell proliferation and induces myogenesis.

In one embodiment, the chemokine receptor is the CCR5 receptor.

In one embodiment, the CCR5 receptor is a tissue stem cell or a tissue stem cell progeny CCR5 receptor. In one embodiment, the CCR5 receptor is a satellite cell or satellite cell progeny CCR5 receptor.

In one embodiment, in vitro, in vivo and ex vivo applications are contemplated.

In one particular embodiment, the present application provides a method of stimulating muscle tissue regeneration, the method comprising administering to muscle an effective amount of a composition comprising or encoding a CCR5 interacting agent, CCR5 interacting agents bind to muscle stem cells (satellite cells) and stimulate myoblast proliferation and muscle regeneration.

In one embodiment, the CCR5 interacting agent is a CCR5 agonist. That is, it stimulates downstream events such as receptor signaling or satellite cell activation and proliferation. In one embodiment, the CCR5 interacting agent specifically activates tissue stem cells. In one embodiment, the CCR5-interacting agent specifically activates satellite cells.

As described herein, in one embodiment tissue regeneration stimulated by the method is associated with minimal fibrosis. Accordingly, in another aspect, the present application provides agents and methods for reducing the incidence of fibrosis in a patient or living tissue subject to regenerative therapy.

In one embodiment, the present application provides methods suitable for regenerating muscle tissue in vitro, in vivo or ex vivo. Accordingly, the CCR5-interacting agents described herein are proposed for use in stem cell-based therapy and tissue engineering. In another embodiment, the CCR5 interacting agents described herein are for use in in vitro artificial meat production.

In one embodiment, the CCR5 interacting agent competes with nicotinamide phosphoribosyltransferase (NAMPT) for interaction with muscle stem cells. NAMPT is a multifunctional protein that, when localized intracellularly, performs well-characterized enzymatic roles in cellular metabolism and NAD regeneration. Also, secretory NAMPT (secNAMPT) functions as a cytokine, but conflicting reports have been made regarding its function in regeneration, physiology, and pathology in many tissues.

In one embodiment, the CCR5 interacting agent capable of promoting stem cell activation is secNAMPT or a portion thereof comprising a cytokine finger (cif) motif or a derivative thereof comprising a cytokine finger (cif) motif. Reference herein to NAMPT includes secNAMPT, which is also a CCR5 interacting molecule as described herein, and derivatives containing portions or fragments thereof.

Accordingly, in one embodiment, the present application provides a method of stimulating tissue regeneration, comprising secNAMPT or a portion thereof comprising a cytokine finger (cif) motif or a derivative thereof comprising a cytokine finger (cif) motif, and Optionally comprising administering to the tissue an effective amount of a composition comprising or encoding a tissue-specific delivery moiety, wherein the secNAMPT, part or derivative binds to adult stem cells in the tissue and activates, proliferates, or activates the stem cells. and stimulates tissue regeneration.

In a further embodiment, the present application provides a method of stimulating muscle tissue regeneration, which method comprises secNAMPT or a portion thereof comprising a cytokine finger (cif) motif or a derivative thereof comprising a cytokine finger (cif) motif or administering to muscle an effective amount of the encoding composition, wherein the secNAMPT, portion or derivative binds to satellite cells and stimulates satellite cell activation, myoblast proliferation and muscle regeneration.

In one embodiment, the present application provides a method of stimulating muscle tissue regeneration, which method comprises secNAMPT or a portion thereof comprising a cytokine finger (cif) motif or a derivative thereof comprising a cytokine finger (cif) motif or administering to muscle an effective amount of the encoding composition, wherein the secNAMPT, portion or derivative binds to satellite cells and stimulates satellite cell activation and myoblast proliferation and muscle regeneration.

In still further embodiments, the present application provides a method of stimulating muscle tissue regeneration, comprising secNAMPT or a portion thereof comprising a cytokine finger (cif) motif or a derivative thereof comprising a cytokine finger (cif) motif and tissue comprising administering to muscle an effective amount of a composition comprising or encoding a specific delivery moiety, wherein the secNAMPT, portion or derivative binds to satellite cells and results in activation of satellite cells, myoblast proliferation and Stimulates muscle regeneration without substantial fibrosis (scarring).

References to NAMPT and CCR5 include homologs and orthologues thereof from any animal, including mammals, non-mammalian vertebrates, fish and birds.

In one embodiment, the NAMPT or portion thereof comprising a cytokine finger (cif) motif or derivative thereof comprising a cytokine finger (cif) motif is an amino acid sequence set forth in any of SEQ ID NOs: 1-4, or at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , contain 99% or 100% identical amino acid sequences.

In one embodiment, the NAMPT portion or fragment thereof comprising a cytokine finger (cif) motif or derivative thereof comprising a cytokine finger (cif) motif is an amino acid sequence set forth in either SEQ ID NO: 1 or 2, or at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% identical amino acid sequences.

In one embodiment, a nucleic acid molecule encoding a full-length NAMPT or functional portion thereof is a polynucleotide sequence set forth in either SEQ ID NO: 8 or 9, or at least 70%, 75%, 80%, 85% , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity comprising a polynucleotide sequence having

In one embodiment, the nucleic acid molecule encoding a NAMPT portion or fragment thereof comprising a cytokine finger (cif) motif or derivative thereof comprising a cytokine finger (cif) motif is a polynucleotide sequence according to either SEQ ID NO: 6 or 7 , or at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% thereof , includes polynucleotide sequences having 97%, 98%, 99% or 100% sequence identity.

In one embodiment, a NAMPT or portion thereof comprising a cytokine finger (cif) motif or derivative thereof comprising a cytokine finger (cif) motif has a few substituted or deleted residues, but is similar to stem cells or satellite cells. retain the cif motif-mediated ability to interact with their more differentiated progeny. In one embodiment, the NAMPT or portion thereof comprising a cytokine finger (cif) motif or derivative thereof comprising a cytokine finger (cif) motif is 1, 2, 3, 4, 5, 6, 7 or Contains amino acid sequences with 8 conservative or non-conservative amino acid substitutions, deletions or additions, but retain CCR5 or tissue stem cell interacting activity. In particular, a functional derivative capable of inducing CCR5 signaling via the Mapk/Erk signaling cascade even when a portion of 1 to 5 contiguous amino acids is deleted from NAMPTcif (SEQ ID NOs: 1, 2, 5). can provide.

In some embodiments, the NAMPT portion or fragment comprises the C-terminal portion of full-length secNAMPT that binds to CCR5 and does not include portions that have enzymatic activity.

In some embodiments, the NAMPT portion or fragment comprises a C-terminal portion of full-length secNAMPT with a chemokine-like α-helix and β-sheet that binds CCR5.

In one embodiment, a NAMPT peptide that does not contain a cif motif can be administered intracellularly to initiate activation of resting muscle cells.

In one embodiment, the NAMPT peptide includes a nicotinamide ribonucleotide binding site (amino acids 219, 384, 392, 311-313, 353-354) or diphosphate binding sites 196, 247, and 311).

In one embodiment, the CCR5 agonist and NAMPT or part or fragment thereof comprise one or more moieties that enhance tissue delivery or retention, such as ECM and/or other tissue-specific binding moieties. While not being bound by a particular mode of action, NAMPT and NAMPT derivatives are particularly selective by binding to target tissue stem cells with their receptors and not other cells with their receptors, thereby can improve the selectivity of

In one embodiment, the CCR5 agonist and NAMPT or part or fragment thereof comprise one or more signaling enhancing moieties such as syndecan binding moieties.

In one embodiment, extracellular matrix (ECM) binding moieties known in the art are included. Exemplary ECM binding peptides are described in US Patent Application Publication No. 2014/0011978 and US Patent Application Publication No. 20140010832. Standard methods are used to conjugate agents or peptides to moieties such as ECM binding moieties, with or without linkers.

In another embodiment, syndecan-binding moieties are included to provide tonicity or enhancement of CCR5 signaling through syndecans.

In one embodiment, the NAMPT portion or fragment is administered in monomeric, dimeric, or multimeric form. In one embodiment, the dimer exhibits increased receptor or tissue stem cell binding compared to the monomeric form.

In one embodiment, the composition is a cell composition comprising cells expressing a CCR5 interacting agent, specifically NAMPT or a functional derivative as described or exemplified herein.

In one embodiment, the present application provides a method of stimulating muscle tissue regeneration comprising in muscle a CCR5 interacting agent and, optionally, a component that enhances muscle delivery or muscle retention. or administering an effective amount of a composition comprising cells encoding, wherein the CCR5 interacting agent binds to satellite cells and stimulates myoblast proliferation and muscle regeneration.

In one embodiment, the cell expresses endogenous NAMPT and/or introduced NAMPT or a functional derivative thereof.

In one embodiment, the cells are macrophages. In one embodiment, macrophages are isolated from tissue. In one embodiment, macrophages are derived from stem cells such as bone marrow progenitor cells or iPSCs. In one embodiment, macrophages or macrophage progenitor cells (monocytes) are isolated from a supplying tissue such as, but not limited to, blood, lymph, bone marrow, and are then isolated to induce a desired tissue niche-tropic phenotype. Finally, it is subjected to in vitro cell or tissue culture. In one embodiment, the cell composition is cryopreserved and/or comprises a delivery agent.

As is known in the art, macrophages can be generated in vitro from stem cells by various means. Macrophages generated from stem cells such as BMSCs in the presence of IFNg or LPS are thought to be 'inflammatory' macrophages, commonly referred to as 'M1 macrophages'. Those produced in the presence of IL-4 and IL-10 have an activity called "pro-resolution" and are called "M2" macrophages.

In one embodiment, the subject's macrophages express the M2 macrophage marker.

In one embodiment, the macrophage cells express one or two or three or four or five of mmp9, arg2, mmp13a, L-plastin and cd163.

In another embodiment, the macrophage subset expresses prox1a and pou2f3.

In one embodiment, the macrophage is a cluster macrophage as described and defined herein, such as cluster 1, cluster 2, cluster 3, cluster 4, cluster 5, cluster 6, cluster 7, or cluster 8 cell types. In one embodiment, the clustered macrophages have the differentiation characteristics described and defined herein prior to culture expansion. In one embodiment, cells are administered in an amount determined by the attending physician, eg, about 1×10 ⁷ or between about 1×10 ⁷ and 2×10 ⁸ cells. The administered cells predominate in one or more cluster types as defined herein. The Examples and Supplementary Tables show differentially expressed genes/markers that can be used for selection, detection, quantification, functional characterization, and discrimination in the predominant macrophage/cell cluster types. Markers may be surface or internal markers, or both, as is known in the art. Thus, markers can be selected based on the level/degree of differential expression to facilitate preferential selection/detection of the most upregulated or downregulated genes. Alternatively, markers and thus cells can be selected based on the functional activity or phenotype of clustered cell types whose detection of the markers indicates desirable/undesirable functional characteristics of the cells. One, few, or multiple markers may be employed, depending on reasons of choice, such as enrichment of cells, selection of cells for administration, tracking of cells, targeting of cells.

In one embodiment, the composition further comprises stem cells and/or macrophage cells.

In one embodiment, the stem cells are satellite cells. In another embodiment, the stem cell is a unipotent or multipotent stem cell.

In one embodiment of this method, the CCR5 interacting agent in the composition is NAMPT or a portion thereof or a functional derivative thereof comprising a cytokine finger (cif) motif, or a pharmaceutically acceptable salt, hydrate thereof , homologues, orthologs, tautomers, sterioisomers, prodrugs.

Prodrug means an agent that can be converted into a CCR5 agonist via some chemical or physiological process (eg, enzymatic processes and metabolic hydrolysis). Accordingly, the term "prodrug" also refers to pharmaceutically acceptable precursors of biologically active compounds. A prodrug is inactive when administered to a subject, but is converted in vivo to an active compound. Prodrug compounds often offer advantages such as solubility, tissue compatibility, or delayed release in vivo. The term "prodrug" may also refer to those containing a covalently bound carrier, such prodrugs releasing the active compound in vivo when administered to a subject. Prodrugs of active compounds can be prepared by modifying functional groups present in the active compound in such a way that they are cleaved, either by routine manipulation or in vivo, into the parent active compound. Prodrugs include any group whose hydroxy, amino or mercapto group is cleaved to form a free hydroxy, amino or mercapto group, respectively, when the prodrug of the active compound is administered to a subject. Included are compounds that bind.

In one embodiment, the CCR5-interacting agent or a portion thereof comprising a NAMPT or cytokine finger (cif) motif or a derivative thereof comprises one or more moieties such as linkers, stability-enhancing, signaling-enhancing, delivery-enhancing or labeling moieties. including.

In one embodiment, the CCR5 interacting agent is in monomeric, dimeric or multimeric form.

In one embodiment, the CCR5-interacting agent or muscle stem cell-interacting agent is NAMPTcif in monomeric, dimeric or multimeric form and also includes tissue-tropic or ECM-binding domains and/or signaling enhancement ( syndecan binding) domains.

In one embodiment, a composition encoding a CCR5 interacting agent comprises a nucleic acid molecule that expresses the CCR5 interacting agent. Nucleic acid molecules can be RNA or DNA or RNA:DNA or chemically modified forms thereof. For example, nucleic acids may be in the form of viral or non-viral vectors.

In another embodiment, the CCR5 interacting agent is a small molecule. The present application provides screening assays for screening small molecule CCR5 agonists capable of inducing tissue stem cell activation. A "small molecule" is defined herein as having a molecular weight of less than or equal to about 1000 Daltons, preferably less than or equal to about 500 Daltons.

In another embodiment, the CCR5 interacting agent is an antibody or comprises a CCR5 binding portion thereof.

In another embodiment, the CCR5-interacting agent comprises an antibody or antibody fragment that targets the agent to myeloid cells, such as macrophages, or targets the agent to stem cells, such as satellite cells.

The present application provides compositions comprising or encoding CCR5 interacting agents as defined herein. Pharmaceutical compositions and physiologically active compositions are provided. A cell composition is expressly provided.

In one embodiment, the cells are macrophages. In one embodiment, macrophages are isolated from tissue. In one embodiment, macrophages are derived from stem cells such as bone marrow progenitor cells or iPSCs. In one embodiment, macrophages or macrophage progenitor cells (monocytes) are isolated from a supplying tissue such as, but not limited to, blood, lymph, bone marrow, and are then isolated to induce a desired tissue niche-tropic phenotype. Finally, it is subjected to in vitro cell or tissue culture. In one embodiment, the cell composition is cryopreserved and/or comprises a delivery agent.

As is known in the art, macrophages can be generated in vitro from stem cells by various means. Macrophages generated from stem cells such as BMSCs in the presence of IFNg or LPS are thought to be 'inflammatory' macrophages, commonly referred to as 'M1 macrophages'. Those produced in the presence of IL-4 and IL-10 have an activity called "pro-resolution" and are called "M2" macrophages.

In one embodiment, the subject's macrophages express the M2 macrophage marker.

In one embodiment, the macrophage cells express one or two or three or four or five of mmp9, arg2, mmp13a, L-plastin and cd163.

In another embodiment, the macrophage subset expresses prox1a and pou2f3.

In one embodiment, the macrophage is a cluster macrophage as described and defined herein, such as cluster 1, cluster 2, cluster 3, cluster 4, cluster 5, cluster 6, cluster 7, or cluster 8 cell types. In one embodiment, the clustered macrophages have the differentiation characteristics described and defined herein prior to culture expansion. In one embodiment, cells are administered in an amount determined by the attending physician, eg, about 1×10 ⁷ or between about 1×10 ⁷ and 2×10 ⁸ cells. The administered cells predominate in one or more cluster types as defined herein. The Examples and Supplementary Tables show differentially expressed genes/markers that can be used for selection, detection, quantification, functional characterization, and discrimination in the predominant macrophage/cell cluster types. Markers may be surface or internal markers, or both, as is known in the art. Thus, markers can be selected based on the level/degree of differential expression to facilitate preferential selection/detection of the most upregulated or downregulated genes. Alternatively, markers and thus cells can be selected based on the functional activity or phenotype of clustered cell types whose detection of the markers indicates desirable/undesirable functional characteristics of the cells. One, few, or multiple markers may be employed, depending on reasons of choice, such as enrichment of cells, selection of cells for administration, tracking of cells, targeting of cells.

In one embodiment, the composition comprises or is administered with a support material such as a hydrogel, adhesive, foam or retainer, scaffold, and the like. Delicate structures are generally preferred to allow regeneration of more delicate tissues. By way of example, very rapidly resorbing materials can be used, such as certain types of fibrin, collagen, hydrogels, alginate formulations. Alternatively, slowly absorbed synthetics such as poly-4-hydroxybutarate can be used. Substantially smooth products of mammalian origin, such as silk fibers, or muscle extracellular matrix are also contemplated. Nonabsorbent composites such as polypropylene and polyethylene provide support and reliability. In one embodiment, the composition comprises fibrin hydrogel. In another embodiment, RAFT-acrylamide-based support surfaces are provided for tissue regeneration and enhanced bioavailability of CCR5 interacting agents to target sites.

In one embodiment, the application provides a NAMPT polypeptide fragment comprising the C-terminal portion of NAMPT comprising a cytokine finger (cif) motif, or a modified form or functional derivative thereof.

In one embodiment, the NAMPT polypeptide fragment is a peptide sequence set forth in SEQ ID NO: 1 or 2 or 5 (monomeric NAMPTcif) or a sequence having at least 80% identity to SEQ ID NO: 1 or 2 or 5 including.

In one embodiment, a NAMPT polypeptide fragment consisting of a peptide sequence set forth in SEQ ID NO: 1 or 2 or 5 (monomeric NAMPTcif) or a sequence having at least 80% identity to SEQ ID NO: 1 or 2 or 5 Encoding nucleic acid molecules are provided. Exemplary nucleic acid sequences are set forth in SEQ ID NOs:6-9 and Table 4. In one embodiment, the nucleic acid molecule has at least 80% identity to SEQ ID NO: 6 or 7 (murine and human NAMPTcif, respectively).

As used herein, at least 80% sequence identity refers to at least 81%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Molecules with 95%, 96%, 97%, 98%, 99% or 100% sequence identity are expressly included.

In one embodiment, the NAMPT fragment or portion is 10%, 15%, 20%, 25%, 30%, 35%, 40% 50%, 60%, 70% of the full-length NAMPT shown in SEQ ID NO:3 , 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or Contains less than 100% of NAMPT amino acids.

In one embodiment, the NAMPT polypeptide fragment further comprises a linker, stability or signaling enhancing moiety, delivery or retention enhancing moiety, or labeling moiety.

In one embodiment, the NAMPT fragment is in monomeric, dimeric or multimeric form. Protein refolding protocols are known in the art.

In one embodiment, a composition is provided comprising a NAMPT polypeptide portion or fragment described herein and any one or two or three or four of: (i) a satellite cell or its (ii) a macrophage or its progenitor cell or its progeny; (iii) a scaffold (semi-solid or solid support) or retention material; (iv) a tissue delivery-enhancing or cell retention moiety.

In one embodiment, the scaffolding or holding material is fibrin or a hydrogel such as an acrylamide hydrogel. In one embodiment, the tissue delivery-enhancing or cell retention moiety is an ECM binding moiety.

In one embodiment, the present application allows for a pharmaceutical or physiologically active regenerating composition comprising 1 or 2 or 3 or 4 or 5 of:
(i) comprising or encoding a CCR5 interacting agent as defined herein (ii) a satellite cell or its progenitor or its progeny (iii) a macrophage or its progenitor or its progeny (iv) a scaffold or Retention material (v) tissue delivery enhancing component.

In certain embodiments, the CCR5-interacting agent in the form of full-length NAMPT, or a cytokine-interacting fragment (cif) thereof in monomeric or dimeric form, as described elsewhere herein is , have been modified to be agents suitable for attachment to biological carriers or extracellular matrices. In addition, the drug has been modified to enhance signaling through the CCR5 receptor by adding co-receptor binding moieties such as heparin sulfate proteoglycans (such as syndecans).

Although CCR5-interacting agents, including proposed modifications, are exemplified in the examples with NAMPT, the experiments described herein demonstrate that the CCR5 agonists CCL4 and CCL8 also act in skeletal muscle satellite cells. It is shown to stimulate proliferation of muscle myoblasts. Accordingly, CCL4 and CCL8 are further exemplary CCR5 agents, full length, portions and fragments (including the cif motif), and derivatives thereof, for use in the methods and of the regenerating compositions described herein. Suggested for use in manufacturing. In certain embodiments, a CCR5-interacting agent in the form of full-length CCL4 or CCL8, or a cytokine-interacting fragment (cif) thereof in monomeric or dimeric form, as described elsewhere herein have been modified to be agents suitable for attachment to biological carriers or to the extracellular matrix. In addition, the drug has been modified to enhance signaling through the CCR5 receptor by adding co-receptor binding moieties such as heparin sulfate proteoglycans (such as syndecans).

In one embodiment, the compositions described herein are for use in stimulating muscle regeneration in vivo, ex vivo or in vitro, or for use in preparing a composition for use. be.

In one embodiment, the compositions described herein are for or are used in artificial muscle production (such as fish, bird or other non-human animal muscle for direct or indirect consumption) It is a matter of time. For example, supplementing growth media with NAMPT allows for scalability and more efficient muscle growth.

In one embodiment, the compositions described herein are for or when used in stem cell therapy. Accordingly, the composition is included during implantation (or as a pretreatment) to support expansion in vitro and/or promote expansion and tissue integration in vivo.

In one embodiment, the present application provides a method of stimulating tissue regeneration, comprising treating isolated or tissue-resident tissue stem cells or their progenitor cells with a CCR5-interacting agent, and optionally to a tissue. wherein the CCR5 interacting agent binds to tissue stem cells or their progenitor cells and stimulates quiescent tissue stem cell proliferation and tissue regeneration ( It is a method of activating). In one embodiment, the CCR5 interacting agent comprises a component or moiety that enhances delivery to target tissue.

In one embodiment, the compositions described herein are for, at, or for use in the treatment of a muscle, neuromuscular, or musculoskeletal defect, disorder, or injury. A composition for use in the manufacture of a product. Muscular, neuromuscular, or musculoskeletal defects, disorders, or injuries are known in the art. Defects and disorders are found, for example, but not limited to, sarcopenia, cachexia and muscular dystrophy, muscle atrophy, muscle pseudohypertrophy or muscular dystrophic conditions and myopathy. All suitable forms are encompassed, such as Swiss-type use, method of treatment and/or EPC2000 form claims.

The patent or application document contains at least one drawing executed in color. Copies of color drawing(s) of this patent or patent application publication will be provided by the Office upon request and payment of the necessary fee.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the invention. be.

A subset of injury-responsive macrophages is present at the wound site during repair. a–c, Intact muscle [Tg(actc1b:GFP), magenta] is patrolled by macrophages (MΦ) [Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), yellow] at 4 dpf (a). Upon injury, MΦs are activated and rapidly migrate to the wound site (bb″, arrows) (n=3). , (0,0) is set as the center of the wound.df, two MΦ subsets occupying the wound at different temporal phases by global imaging (f), the transient subset responding to early injury (d ) and a resident subset (e) (arrows) at the site of late injury were identified (n=8) g–i, morphological characterization of these two subsets revealed that the transient MΦ was more stellar The morphology was found to be (g) less sphericity (i), the resident MΦ more round (h) and more sphericity (i) (n=69 MΦs in each group).mean±s.d. Deviation Significance in unpaired t-test ( ^*** P<0.0001) jm, from follow-up of MΦs located at the wound site, resident MΦs are derived from transient MΦs (j) Schematic diagram of the photoconversion strategy: Photoconversion of the damaged site MΦ of Tg (mpeg1:Gal4FF/UAS:Kaede) larvae at 1 dpi (kk'', before conversion: magenta (arrow ), after conversion: yellow), re-evaluated at 2 dpi (l−l″) and quantified (m, n=20). Unpaired t-test (statistically) not significantly different (ns). Retrospective tracing of the resident MΦ present in the nn′, 24 hpi lesion identified its near-injury origin. (n) Track projections of the resident MΦ during the imaging period were superimposed over the entire larva. (n′) Location of resident MΦs (yellow) at different time points after imaging, showing their origin and sequential migration (blue) to the injury site (n=4). Resident macrophages induce proliferation of muscle stem cells in vivo. a, Schematic of dosing strategy for addition of prodrug metronidazole (Mtz) to ablate all MΦs or MΦs specifically present in Tg (mpeg1:GAL4FF/UAS:NfsB-mCherry) larvae. bh, both of these ablation strategies result in significant regeneration defects as observed by birefringence imaging (bg′) and quantification (h, n=24). il, After laser ablation injury (dotted line), transient MΦs migrated to the wound site [Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), yellow] (ik). At the same time, activated pax3a ⁺ myogenic stem cells [TgBAC(pax3a:GFP), cyan] from myogenic areas adjacent to the injury site migrate to line the edges of the injury site (ik, arrows). As MΦs transition to the resident subtype, they begin interacting with pax3a ⁺ cells at the wound margin (l, orthogonal view) (n=5). m, Length of continuous interaction between transient and resident MΦ macrophages and stem cells quantified (extracted from n=5 lesions). Mean ± standard deviation. Unpaired t-test. n, High-resolution AiryScan microscopy of these interactions revealed that resident MΦs remained in tight association with pax3a ⁺ muscle stem cells (arrows) for extended periods of time, after which the associated stem cells underwent cell division. (n=10). oq, EdU incorporation assessment o) of stem cell proliferation after resident MΦ ablation allowed us to confirm the requirement of this MΦ subset for the maintenance of muscle stem cell division at the wound site. All EdU ⁺ proliferating cells at the injury site were co-labeled with pax3a, revealing that myogenic stem/progenitor cells were the only proliferating cells involved in regeneration at this point in the repair process. rice field. The homeostatic level of myogenic cell proliferation outside the injured area provides an internal specificity control for these analyses, and no significant difference in cell proliferation with or without resident MΦ is observed outside the wound. Quantification (q). Mean ± standard deviation. Significance in two-way ANOVA with Tukey's multiple comparison test (***P<0.0001). Single-cell RNA-seq identifies a unique mmp9-positive resident macrophage subset. a, Schematic of injury-responsive macrophage scRNA-seq workflow. b, UMAP scatter plot revealed spontaneous cell clusters. After appropriate filtering, 1309 cells were analyzed. c, In this graph, isolated MΦ injury time points are superimposed on a UMAP scatterplot showing unsupervised clustering and correspondence between transient and resident MΦ subtypes described. ing. d, Intact MΦ cluster together (cluster 3). Macrophages isolated from the 'transient' time point (1 dpi) also cluster predominantly (cluster 1). The remaining 6 clusters (0, 2, 4, 5, 6, 7) are composed primarily of macrophages isolated from 'resident' time points (2-3 dpi). e, Macrophage identity of sorted cells was verified by collective expression of pan-leukocyte marker lcp1 (L-plastin) and pan-macrophage marker cd163 (cd63). The known pro-regenerative MΦ markers arg2, mmp9, mmp13a are clustered in the MΦs present in cluster 2. The percentage of cluster 2 cells expressing these markers is shown. In addition, this cluster also differentially expresses nampta. Gene expression levels visualized in feature plot lines are log-normalized gene read counts using a scaling factor of 10,000. f, Violin plots show the expression pattern of mmp9 and nampta based on cluster identity and isolation time point. Record the percentage of cells expressing the marker at 2 dpi. gk. Phylogenetic analysis was performed using partition-based graph abstraction (PAGA). Ball-and-stick representation of PAGA binding; pie charts represent clusters (size reflecting cluster cell size) showing macrophage composition based on isolation time; edge thickness is statistics of connectivity between clusters. (g). Cells were re-embedded using PAGA initialization Force Direct Layout (FDL), where cells were grouped according to their isolation time point identity (h) Seurat cluster identity (i) and pseudo-temporal inference (j). turned into (k) PAGA path graph shows mmp9 ⁺ - resident macrophage subsets along path 3-0-4-2 of PAGA clusters and prox1a ⁺ /pou2f3 ⁺ - along path 3-5-1-6 of PAGA clusters. Visualization of gene expression changes leading to the definition of resident macrophage subsets. The numerical scale (right) represents Seurat-normalized gene expression, while the clusters (bottom) indicate subsets along the PAGA pathway, with length proportional to the number of cells in each cluster. l. Spatiotemporal expression of mmp9 RNA after needlestick injury was assayed by in situ hybridization. Expression of mmp9 is specifically upregulated at the wound site after 2 dpi (n>15). mn, Spatially resolved mmp9 expression [TgBAC(mmp9:EGFP), magenta] at the wound site and a subset of resident MΦs [Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), yellow] ( White arrow) was confirmed to exist. Quantitation (n). Mean ± standard deviation. Unpaired t-test. (o) These mmp9-expressing MΦs (magenta) were associated with wound-resident pax3a ⁺ stem cells (cyan, wound site: white dotted line, n=15). pr, Temporarily controlled metronidazole (Mtz)-based ablation of mmp9-expressing resident MΦs of TgBAC(mmp9:EGFP-NTR) larvae resulted in marked skeletal muscle regeneration defects (p). Quantification (q). Assaying EdU uptake in mmp9 ⁺ MΦ post-ablation larvae revealed significantly reduced proliferation of muscle stem cells located at the wound site (r). A representative image is shown in FIG. 10g. Mean ± standard deviation. Two-way ANOVA (q, r) with Tukey's multiple comparison test.

When Nampt secreted from macrophages binds to Ccr5, proliferation of stem cells is induced. a, Within the injury site (white dotted line), Nampt (magenta) is expressed by macrophages (yellow, red arrows highlight Nampt ⁺ /mpeg1 ⁺ MΦs) associated with muscle stem cells (cyan) and is also present in the extracellular space. (n=15). b–i, A novel tissue-specific loss-of-function mutagenesis strategy was utilized to identify the specific roles of Nampta in injury-responsive macrophages (schematic, b) and Ccr5 in muscle stem/progenitor cells (schematic). , c) were evaluated independently. Both Mpeg ⁺ cell-specific Nampta and Pax7b ⁺ cell-specific Ccr5 knockout larvae showed marked regeneration failure [imaging (d) and quantification (e)]. In both cases, the inability to sustain the required proliferative response in wound-resident muscle stem cells [images (f,h) and quantifications (g,i)] was the cause. j, NAMPT supplementation specifically enhanced proliferation of wound-resident muscle stem cells in zebrafish needleprick muscle-injured larvae. Supplementation with NAMPT rescues the proliferation defect caused by macrophage ablation (4 dpf/0 dpi plus 5 mM Mtz). However, NAMPT was unable to rescue the growth defect caused by administration of the Ccr5/Ccr2 dual antagonist Senicriviroc (CVC). The Ccr5 ligand, CCL4, has the function of enhancing proliferation, but failed to distinguish between injury and extra-injury sites. Probability values and significant differences for experimental conditions versus untreated controls were recorded above each violin plot. A representative image is shown in Figure 14g. k, PAX7 ⁺ satellite cells in mouse primary myoblast monocultures show enhanced proliferation by CCR5 receptor signaling mediated by either exogenous NAMPT or CCL4 supplementation. Co-culturing myoblasts with macrophages enhanced the proliferation of satellite cells. This proliferation-promoting response was abolished by administration of the CCR5-specific inhibitor maraviroc (MVC), and the CCR2-specific inhibitor PF-4136309 (PF4) did not show a negative effect, indicating that the proliferation-promoting function of macrophages on stem cells was due to CCR5 It was reconfirmed that it is mediated by signal transduction. In addition, co-culturing myoblasts with 3T3 cells that do not secrete NAMPT does not promote satellite cell proliferation, suggesting that NAMPT is a macrophage-driven proliferation-promoting cue that promotes stem cell proliferation. e, g, i, j, k, mean±s.d. Two-way ANOVA with Tukey's multiple comparison test. lo, Local delivery of NAMPT promotes muscle regeneration in an adult mouse muscle injury model (schematic, l). (m) A volumetric muscle defect was created and treated directly with NAMPT delivered via fibrin hydrogel. Representative tissue sections of mouse rectus femoris (RF) muscle (10 days after treatment) were stained with Masson's trichrome through the center of the defect and NAMPT delivery correlated with regenerated muscle area (dark red, quantification, n ) while significantly increasing fibrous tissue [purple/blue, white dashed line demarcates separation between fibrous and healthy muscle fibers, perimuscular fascia stained blue (quantification, o)] showed a significant decrease. n, o, mean±s.d. One-way ANOVA and Dunnett's post hoc test for multiple comparisons (n=5 mice/group). pt, Satellite cells showed enhanced proliferation with exogenous NAMPT supplementation. Mouse muscle lesions were treated with NAMPT (0.5 μg) delivered in fibrin or a fibrin-only control. (p-q) Total number of satellite cells (PAX7 ⁺ ) (p) and number of proliferating satellite cells (PAX7 ⁺ /Ki67 ⁺ ) (q) in tissues harvested 4 days after treatment by flow cytometry. quantified. The graph shows the ratio of satellite cells per 10,000 cells in the collected tissue (fibrin-administered group: n=6 mice, NAMPT-administered group: n=5 mice). The gating strategy is shown in Figure 10e. (r) Representative muscle regeneration cryosections stained for PAX7 (satellite cells, yellow), wheat germ agglutinin (WGA, magenta), and nuclei (DAPI, blue) from tissue taken 6 days after treatment. . (s to t) Central nucleated muscle fibers were quantified 6 days after administration (n=6 mice per group) (s). Representative tissue sections (t) by hematoxylin and eosin. p, q, s, mean ± S.E.M. E. M. . Two-tailed Student's t-test. Resident macrophages form a transient niche that is essential for the regeneration process after muscle injury. a–c″, maximum intensity projection images of the same fish, after pinprick muscle injury, Tg(actc1:BFP) labels differentiated muscle fibers [magenta (a′,b′,c′)], TgBAC(pax3a:GFP ) labels pax3a ⁺ myogenic cells [cyan (a'', b'', c'')], and Tg (mpeg1: GAL4FF/UAS: NfsB-mCherry) labels macrophages [yellow (a'' , b''', c''')]. d, 51.6% of the original injury-responsive MΦs remain at the wound site by 2 dpi, and these resident MΦs continue to establish transient injury niches that are myogenic (n=20). ei, Establishment of nitroreductase-mediated macrophage ablation parameters. (ei) A transgenic line, Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), specifically expresses the enzyme nitroreductor in macrophages and the addition of the prodrug metronidazole (Mtz) results in macrophage time allows for systematically controlled genetic ablation. Optimal Mtz concentrations were established by treating larvae with 5 and 10 mM Mtz for 24 h (from 4 dpf) and visualizing (e) and quantifying (f) MΦs present in the trunk. 5 mM Mtz treatment achieved 77% MΦ ablation at 5 dpf (f, n=10). Intact larvae were able to tolerate 10 mM Mtz, but needlestick injuries performed at this concentration resulted in significant lethality. Therefore, 5 mM Mtz was used for MΦ ablation in all subsequent experiments. (gg') Assay of Mtz dosing time to specifically ablate the MΦ subset of targets. (g) Temporal responses to 5 mM Mtz were visualized by time series imaging of head and tail regions of larval zebrafish immediately after Mtz addition (4 dpf). Ablation of MΦ begins 3 hours after Mtz treatment. From additional video 5, extract frames (n=8). (g′) Addition of Mtz at the time of injury ablated all MΦs (yellow, superimposed on bright-field image of zebrafish trunk), while addition of Mtz at 1.75 dpi (5 dpf) Resident MΦs were selectively ablated (n=10). (hi) Ablation of macrophages did not alter neutrophil responses to wounding at 2 dpi [imaging (h) and quantification (i), n=12]. j, Temporal sequence of neutrophil migration in response to skeletal muscle injury was assayed and quantified (k, n=14 at 5-6 hpi, n=13 at 1 dpi and n=12 at 2 dpi). lo, As a control experiment for nitroreductase-mediated cell ablation, neutrophils were selectively ablated using the transgenic line Tg (mpx:KALTA4/UAS:NfsB-mCherry). Starting at 4 dpf, ablation efficiency after 24 h of 5 mM Mtz treatment was visualized (l) and quantified at 5 dpf (m, n=10). (n) After needlestick injury, larvae were bathed in 5 mM and 10 mM Mtz (4 dpf/0 dpi) to ablate all neutrophils and 5 dpf (1.75 dpi) to bathe in 5 mM Mtz to specifically target neutrophils late in the injury. Ablation (continuing Mtz treatment with daily Mtz drug renewal until the experimental endpoint of 7 dpf/3 dpi) and birefringence imaging to monitor regeneration (n) and quantify (o, n=12). Neutrophil-ablated larvae do not exhibit regeneration defects at the timing of administration of Mtz, thus ruling out the specific requirement of macrophages in stimulating regeneration and any possibility of regeneration defects for metronidazole. emphasized that d, f, k, m, mean±SEM. D. . One-way ANOVA with Dunnett's post-hoc test. i, mean ± S.E.M. D. . Unpaired t-test. o, mean ± S.E.M. D. . Two-way ANOVA with Tukey's multiple comparison test.

Ablation of macrophages does not prevent clearance of tissue debris after muscle injury. af, Macrophages within a transgenic line of Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry) (nitroreductase-expressing macrophages) at 3 dpf were ablated from zebrafish larvae by adding 5 mM metronidazole (Mtz). bottom. Macrophage-ablated larvae were subjected to needle-prick skeletal muscle injury at 4 dpf and the wound site was assayed for clearance of injured muscle fibers by several criteria. (a) Phalloidin staining, showing residual muscle fibers. No difference is seen between control larvae and macrophage ablated larvae. n=3 independent lesions were shown at each time point and n=12 lesions were imaged at each time point. (b-c) Annexin V demonstrating apoptotic/phagocytic signaling processes was visualized in vivo using the transgenic line Tg (ubi:secAnnexinV-mVenus) (magenta, presenting n=3 independent lesions at each time point). (b), and quantified (c). (d–e) Acidic organelles marked with LysoTracker (magenta) were imaged (d) and quantified (e). (c, e) Mean ± S.E.M. D. . Two-way ANOVA with Tukey's multiple comparison test (c) or unpaired t-test (e). No difference is seen between control larvae and macrophage ablated larvae. (f) Hematoxylin and eosin-stained cross-sections of needlestick-injured myoblasts of macrophage ablation larvae show that the wound site (black arrow) has cleared 3 days after wounding (n=3 larvae per group). Resident macrophage-muscle stem cell association precedes muscle stem cell proliferation. ac, pax3a ⁺ myogenic cells with which macrophages interact also express the muscle stem cell markers met (ab) and pax7b (c). (a) TgBAC(pax3a:GFP) (cyan) and Tg(met:mCherry-2A-KALTA4/UAS:NfsB-mCherry) (magenta) transgenic larvae after needlestick skeletal muscle injury (white dotted line), wound area. The muscle stem cells present in the cells were pax3a ⁺ /met ⁺ . These cells undergo both symmetric expansion (open arrows) and asymmetric division (closed yellow arrows), each resulting in two pax3a ⁺ /met ⁺ daughter cells, or pax3a ⁺ /met ⁺ and pax3a ^+. /met- gives rise to daughter cells. The broad myogenic marker pax3a ⁺ visualizes each of these events (n=20). (b) muscle stem cells [TgBAC(pax3a:GFP)/Tg(mCherry-2A-KALTA4/UAS:NfsB-mCherry), cyan/yellow double positive cells] and endogenous macrophages [Tg(mpeg1:GAL4FF/UAS:NfsB -mCherry), yellow] interactions following laser ablation skeletal muscle injury (dotted white line), time series imaging was used. Resident macrophages (white closed arrows) interact with pax3a ⁺ /met ⁺ muscle stem cells (magenta open arrows), which then undergo symmetric division to form two pax3a ⁺ /met cells. ⁺ to produce a precursor. From additional video 9, extract frames (n=6). (c) Resident macrophages [Tg (mpeg1: GFP), yellow] present at the wound site (dotted white line) are muscle stem cells [cyan, Tg (pax7b: GAL4FF/UAS: NfsB-mCherry) that also express the marker pax7b ]. df, Muscle stem cells migrate to the injury site independently of macrophage-derived signals. Macrophages [Tg (mpeg1: GAL4FF/UAS: NfsB-mCherry), yellow] and muscle stem cells [TgBAC (pax3a: GFP), cyan] migrate and migrate to the injured site at the same time, so the dependence of one on the other is evaluated. bottom. Pax3a ⁺ stem cells (white dotted line) were present at the site of injury at 2 dpi (white arrow) after needlestick skeletal muscle injury in both control and macrophage-ablated larvae [all macrophages were ablated by adding 5 mM Mtz at the time of injury (4 dpf)]. (d), and muscle stem cells were confirmed to line the injury site edge (yellow asterisk) after 14 hpi laser ablation muscle injury (e). Quantification of macrophages in response to laser injury (f). Mean ± standard deviation. No significant difference by unpaired t-test. In the control setting (top d, e), these cells were associated with macrophages. At 3 dpi after needlestick injury, control larvae show regenerated cyan fluorescent persistent muscle fibers (red arrows) originating from pax3a ⁺ muscle stem cells present in the wound (d), which is characteristic of muscle injury healing. . On the other hand, in macrophage-depleted larvae, the wound site was still occupied by pax3a ⁺ stem cells at 3 dpi, but pax3a ⁺ stem cell-derived new myofibers were absent (d), indicating that the wound site We emphasize that the presence of macrophages, although not essential for the migration of pax3a ⁺ myogenic cells, is essential for those migrated cells to enter the myogenic recruitment program (n=12). g, Resident macrophages interact with wound-responsive muscle stem cells. Three independent examples of laser ablation muscle injury sites show binding of resident macrophages to stem cells followed by stem cell division (arrows). In transgenic zebrafish larvae, Tg (mpeg1: GAL4FF/UAS: NfsB-mCherry) labeled macrophages (yellow) and TgBAC (pax3a: GFP) labeled pax3a ⁺ cells (cyan) for imaging. Extract frames from additional video 7. h, After muscle stem cell division, associated resident macrophages and generated daughter cells migrate away from each other. Resident macrophages interact with muscle stem cells present at the injury site (dotted white line). After muscle stem cell division (closed white, magenta, yellow, gray arrows highlight four independent stem cells), daughter cells (open white, magenta, yellow, gray arrows are closed arrows of the same color) Emphasize daughter cells generated from stem cells shown in ) movement within the wound site to visualize their relationship with endogenous macrophages. Cell movements are followed until resident macrophages become localized in the vertical sarcomere septum. Extract frames from additional video 10. A representative example of n=9 stem cell divisions assayed over n=5 long time courses. Correlative light-electron microscopy (CLEM) analysis of the transient macrophage-stem cell niche revealed that macrophages and stem cells maintain direct heterologous cell surface attachment in the xyz plane. a, Confocal micrograph of wound site 25 hours after muscle laser ablation injury, multigene zebrafish strain, Tg (mpeg1: GAL4FF/UAS: NfsB-mCherry), TgBAC (pax3a: GFP), macrophages (yellow). and labeled with pax3a ⁺ muscle trunk/progenitor (cyan). b, Large STEM tileset after Epon embedding and sectioning of the same larval trunk shown in (a). This dataset was used to correlate and identify highlighted macrophage-stem cell interactions [white asterisks (a,b)]. Dotted squares are further examined by transmission electron microscopy (c, d). c, Target area encompassing macrophages and stem cells maintaining close interactions examined by z-depth. Divide cells in target and interaction areas (macrophage cytoplasm: yellow, macrophage nucleus: orange, stem cell cytoplasm: cyan, stem cell nucleus: blue, cell-cell interaction surface: magenta). High-resolution images of the two planes through d,z depth (planes shown correlated with red dotted lines in the segmentation image) further show the close association of the two cells (black scale bar: 5 μm). .

Prox1a ⁺ /Pou2f3 ⁺ macrophages (cluster 6) identified by scRNA-seq are a subset of host macrophages independent of Mmp9 ⁺ macrophages (cluster 2). a, Cells assayed by scRNA-seq expressed antigen-treated and presented genes, confirming macrophage identity. Feature plots highlighting antigen-presenting genes (cd83, cd81a, and cd40) expressed in the majority of cells and differential expression by individual subsets are shown. Also record the percentage of cells expressing the gene of interest (maroon, information extracted from Supplementary Tables 1, 3). bg, Violin plots (percentage of cells in cluster 6 expressing markers recorded as a percentage (b,e) and specific markers Pou2f3 (c) and Prox1a (f) of subset 6 after needlestick injury) Antibody staining for mCherry (white dotted line) identifies a specific post-injury resident macrophage population (Supplementary Table 3, Worksheet 7) Antibody staining for mCherry identifies Tg(mpeg1:GAL4FF/UAS:Nfsb- mCherry) (yellow) lineage, Few Pou2f3 ^{+ /} mpeg ⁺ (c) or Prox1a ⁺ /mpeg ⁺ (f) macrophages are present at the wound site at 1 dpi after injury. Their number started to increase slightly at 2 dpi and the highest proportion was present at 3 dpi, highlighting that this subset is the resident macrophage subset in the late stage of injury.Quantification (d,g).Mean±s.d. (d) One-way ANOVA with Dunnett's post-hoc test (g) hi, cluster 2 macrophages show a metabolic shift towards glycolysis at the gene expression level of cluster 2 KEGG pathway analysis of differentially expressed genes (Supplementary Table 3) identified genes associated with the glycolysis (term: dre00010) and oxidative phosphorylation (term: dre00190) pathways. was upregulated (h), while most of the genes associated with oxidative phosphorylation were downregulated (i), highlighting the metabolic shift towards glycolysis in cluster 2/mmp9 ⁺ -resident macrophages. Figure 9. a NAMPT contains a conserved "cytokine finger" (cif) in other cytokines b NAMPTcif inhibits binding of NAMPT to CCR5 c NAMPTcif in satellite cells stimulation dose-dependently promotes proliferation compared to a non-stimulated control baseline (Nostim) d.Derivatives of NAMPT with or without ECM and syndecan-binding tags are tested in vivo and in vitro. The intracellular enzymatic function of NAMPT does not govern its growth-promoting function in skeletal muscle regeneration. ab, mmp9 marks a subset of wound-resident macrophages. (a) After laser-induced skeletal muscle injury (dotted white line), a subset of macrophages [Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), yellow] resident mmp9 [TgBAC(mmp9:EGFP), magenta]. Begins to express (yellow/magenta double labeled cells). After injury, mpeg ⁺ macrophages respond to wounding. At the same time, mmp9-expressing neutrophils are also found to be present at the injury site (magenta only, green open arrows, showing cell phenotypes and wound site dynamics distinct from macrophages. These cells are neutrophilic (data not shown, n=16) These mmp9-expressing neutrophils exit the wound site by 5.21±1.27 hpi (n=6). ). As macrophages become resident, a subset of mpeg-expressing macrophages also begin to express mmp9 (white closed arrows). From additional video 10, extract frames (n=6). (b) Titrating metronidazole (Mtz) dose allows specific ablation of mmp9-expressing immune cells in TgBAC(mmp9:EGF-NTR) (magenta) transgenic lines. Upon treatment with 5 mM Mtz for 6 h [1.75 dpi (5 dpf) to 2 dpi (6 dpf)], intact larvae showed ablation of mmp9-expressing immune cells (phenotypically distinct, stellate morphology, high mmp9-expressing cells). , whereas skin cells with significantly lower levels of mmp9 expression (phenotypically distinct, hexagonal morphology) were shown to be resistant to ablation. Larvae treated with Mtz at 5 dpf/1.75 dpi after needlestick skeletal muscle injury (4 dpf/0 dpi) showed a lack of mmp9-high expressing cells (n=12), especially at the wound site (white dotted line). cd, The dose and duration of Mtz treatment used specifically ablate mmp9 ⁺ /mpeg ⁺ macrophages by 77.94% (mmp9−/mpeg ⁺ macrophages: 6.65±1.50 with Mtz treatment vs control 5.47±2.75 in mmp9 ⁺ /mpeg ⁺ macrophages: 2.18±1.19 in Mtz-treated vs. 9.87±2.72 in controls). (c) Representative image. mmp9 ⁺ /mpeg ⁺ (magenta and yellow, respectively) macrophages within the needleprick skeletal muscle injury (white dotted line) are identified by white arrows. (d) Quantification. eg, Ablation of mmp9 ⁺ macrophages results in a marked reduction of muscle stem/progenitor cells present at the wound site. There is a marked reduction in Pax7 ⁺ muscle stem cells present at the wound site (white dotted line) after Mtz treatment (e). (f) Quantification. This reduction was not due to a defect in stem cell migration, as Mtz treatment follows muscle stem cell migration (muscle stem cells present at the wound margin of Mtz-treated larvae can be visualized in e), but rather EdU uptake. (g, shows three independent cases of larval injury in both control and Mtz-treated larvae due to defective proliferation of muscle stem cells within the wound site (yellow dotted line) observed by measuring the representative of the quantifications shown). d, f, mean ± S.E.M. D. . Unpaired t-test. h, nampta mRNA expression is specifically upregulated at the injury site after 2 dpi in zebrafish larvae (black arrows, n>15). ij, Increased NAMPT activity leads to elevated intracellular NADH levels. (i) NADH autofluorescence (magenta) was locally elevated in resident macrophages (yellow), indicating that these cells were the major source of Nampt present in the wound (n=15). ). (j) This was further quantitatively confirmed by a bioluminescence-based assay, showing that resident MΦs located in wounds have a lower NAD ⁺ /NADH ratio compared to transient MΦs. rice field. Mean ± standard deviation. Unpaired t-test. ko, Evaluation of Nampt expression in larval zebrafish. Nampt antibody staining performed on developmental lines (1, 3, 4 dpf) confirmed the agreement of the protein distribution pattern with previously reported RNA transcripts ³² , indicating ubiquitous expression early in development and at somite boundaries at 1–4 dpf. (white closed arrows) and further concentrated in the intestine (white open arrows) after 3 dpf (n=10) (k). (lm) Wound site Nampt protein expression is upregulated from 6 dpf/2 dpi after needlestick skeletal muscle injury. Quantification (m). Mean ± standard deviation. Unpaired t-test. (no) Wound site Nampt expression elevation (white dotted line) is derived from macrophages, and selective ablation of macrophages [5 mM Mtz at the time of injury (4 dpf/0 dpi) Tg (mpeg1: GAL4FF/UAS: NfsB -mCherry) added to larvae to ablate all macrophages], wound site Nampt expression was markedly reduced, indicating that it was of macrophage origin. This is further confirmed by the fact that selective ablation of neutrophils [Tg(mpx:KALTA4/UAS:NfsB-mCherry) larvae used for neutrophil ablation] does not perturb Nampt levels either. Quantification (o). Mean ± standard deviation. Two-way ANOVA with Tukey's multiple comparison test. Inhibition of enzymatic function of pq, Nampt does not affect muscle stem cell proliferation. After needlestick skeletal muscle injury (dotted white line), TgBAC(pax3a:GFP) (cyan) larvae were treated with the small molecule competitive inhibitor GMX1778 from 5 dpf/1.75 dpi to the end of the experiment (6 dpf/2.5-2.75 dpi). treated up to GMX1778 selectively inhibited the rate-limiting enzymatic function of Nampt in NAD ⁺ biosynthesis and markedly reduced larval NAD ⁺ /NADH levels at dosed concentrations (see FIG. 10c). Inhibition of Nampt's enzymatic function had no effect on muscle stem cell proliferation at the site of injury (p), highlighting that Nampt's function during stem cell proliferation is distinct from its intracellular role in energy metabolism. . Quantification (q). Mean ± standard deviation. Two-way ANOVA with Tukey's multiple comparison test.

NAMPT binds to the CCR5 receptor present on muscle stem cells and induces proliferation. ab, NAMPT selectively binds to CCR5. (a) ELISA plates were coated with human recombinant CCR5 (hrCCR5) or BSA and incubated with human recombinant NAMPT(1) [hrNAMPT(1)] at increasing concentrations (0 nM to 800 nM). NAMPT molecules bound to CCR5 were detected via a biotinylated antibody. NAMPT bound to CCR5 with a K _D of 172±18 nM (n=2 independent experiments, triplicate). The graph shows a representative binding curve with non-specific binding to BSA subtracted. Mean ± S.E.M. E. M. . (b) ELISA plates were coated with mouse recombinant CCR5 (mrCCR5) or BSA and incubated with increasing concentrations (0 nM-400 nM) of mouse recombinant CCL4 (mrCCL4) along with 100 nM hrNAMPT ₍₁₎ . hrNAMPT ₍₁₎ molecules bound to CCR5 were detected via a biotinylated antibody. mrCCL4 showed an _IC50 of 34.4±2.2 nM (n=2 independent replicates). The graph shows binding curves with non-specific binding to BSA subtracted. Data are mean±SEM. c, Cultured Maf/DKO macrophages actively secrete NAMPT. NAMPT concentrations in the supernatants of Maf/DKO and Raw264.7 macrophages cultured for 16 h were quantified by ELISA (n=1 experiment replicated in triplicate, stimulated with 10 ng/ml M-CSF). Mean ± standard deviation. Unpaired t-test. d-e, Cultured Maf/DKO macrophages do not actively secrete CCL3, CCL4, CCL5, the cognate ligands of CCR5. (d) Representative array spots detecting CCR5 ligand expressed by Maf/DKO macrophages after stimulation with M-CSF (10 ng/ml) for 16 h. (e) Quantification. Mean ± S.E.M. E. M. (n=1 array per group, 2 replicates). f, Exogenous NAMPT supplementation promotes myoblast proliferation. In vitro assay to assess the effect of exogenously introduced factors on proliferation of C2C12 myoblasts. Proliferation is identified by EdU incorporation. Administration of NAMPT [two commercially available NAMPT sources, hrNAMPT ₍₁₎ and hrNAMPT ₍₂₎ ] was shown to increase myoblast proliferation in a dose-dependent manner. This effect is specifically mediated through the CCR5 receptor. Co-administration of the CCR2/CCR5 dual inhibitor senicriviroc (CVC) and the CCR5-specific inhibitor maraviroc (MVC) and NAMPT abolished the growth-promoting response of NAMPT, whereas co-administration with the CCR2 inhibitor PF-4136309 (PF) Administration does not inhibit the myoblast proliferation-stimulating effect of NAMPT. Consistent with this finding, the endogenous ligands of CCR5, mrCCL8 and mrCCL4, functioned to promote proliferation of C2C12, whereas the CCR2-specific ligand, mrCCL2, failed to enhance proliferation rates above controls. . The growth-promoting function of NAMPT was found to be separate from its intracellular role in energy metabolism, as concomitant use of the NAMPT enzyme inhibitor GMX1778 did not affect its effect on myoblast proliferation. Mean ± standard deviation. Two-way ANOVA, Tukey's multiple comparison test. g, NAMPT stimulates proliferation of PAX7 ⁺ satellite cells in mouse primary myoblast monocultures. This growth-promoting response is abolished upon administration of CVC. Co-culture of myoblasts with macrophages promotes satellite cell proliferation, but this response is inhibited by administration of CVC. Co-culture of myoblasts with 3T3 cells that do not secrete NAMPT does not stimulate proliferation of satellite cells. Mean ± standard deviation. Two-way ANOVA and Tukey's multiple comparison test. Inhibition of hp, Ccr5 receptor activation does not affect the dynamics of injury-responsive immune cells in zebrafish larvae. (hi) Zebrafish predicted orthologous Ccr5 gene was identified by using BLAST to determine the appropriate best hit of the human CCR5 amino acid sequence in the zebrafish genome. A maximum likelihood phylogenetic tree constructed using the protein sequence of Ccr5 placed a putative zebrafish Ccr5-like (Ccr5L) sequence as a homologue of mammalian CCR5 with strong bootstrap support. Bootstrap values from 500 replicates are recorded under the phylogenetic tree. CCR7 was used as an outgroup. The accession numbers of the genes included in the analysis are shown in Table (i). (j) pax3a ⁺ muscle stem cells express zfCcr5 receptor at 2 dpi as detected by RT-PCR. (kl) Ccr5 inhibition by the receptor antagonist CVC results in migration of macrophages [Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), yellow] to the site of injury [Tg(actc1b:BFP), scaffolds labeled in magenta. muscle] and good migration into host MΦ subtypes. Quantification (l). (mn) CVC-treated macrophages appear morphologically indistinguishable from controls, with transient MΦs having lower sphericity values than their resident counterparts. Quantification (n). (l, n) Mean±s.d. Two-way ANOVA with Tukey's multiple comparison test. (op) CVC treatment does not alter the neutrophil [Tg(mpx:eGFP), magenta] response to needle-prick muscle injury (dotted white line) (o). Quantification (p). Mean ± standard deviation. Unpaired t-test. q, CVC treatment [2 h pretreatment, maintained until experimental endpoint after laser ablation skeletal muscle injury (white dotted line)] stimulates the initiation and association of resident macrophages (white arrows) and muscle stem cells (white arrows) at the wound site. Do not interfere with maintenance. Extract frames from additional video 13 . Larvae immersed in r to s, CVC or MVC showed marked regeneration failure in birefringence imaging (r) and quantification (s). Proliferation of t to u, pax3a ⁺ myogenic stem cells [TgBAC(pax3a:GFP), cyan] is demonstrated by decreased EdU incorporation (magenta) in these cells after injury (t) and quantification (u). was inhibited by CVC addition, as shown. Two-way ANOVA with Tukey's multiple comparison test. Zebrafish germline nampta and ccr5 mutants exhibit severe skeletal muscle regeneration defects in response to acute muscle injury. aj, Targeting exon 2 of nampta using CRISPR/Cas9 resulted in a germline deletion-insertion mutation, resulting in an amino acid sequence change followed by a premature stop codon (asterisk) (namptp .Try61Profs*4, referred to as nampta ^pc41 ). (a) Sanger traces of genotyping PCR amplicons showing the effects of Cas9/gRNA mutagenesis at the DNA and amino acid sequence (AA) level of the target nampta locus. (b) Schematic representation of the nampta gene, highlighting its exons, mutation sites, transcripts, and primer binding sites for reverse transcriptase (RT) PCR. (c) RT-PCR of the nampt cDNA shows decreased levels of the mutant transcript encoding the truncated protein, highlighting that it is targeted for degradation by nonsense-mediated decay. (df) Macrophage and stem cell dynamics are unaffected in nampta mutants. (d) Both nampta heterozygous and homozygous mutants show comparable dynamics of macrophages (yellow) at the wound site (dotted white line) to wild-type siblings, with macrophages transitioning to a resident state at 2 dpi. (e) Quantification. (f) Furthermore, resident macrophages of heterozygous and homozygous nampta mutants come to interact with Pax7 ⁺ muscle stem cells present at the wound site. Representative example of n=20 observations. Homozygous mutants show severe regenerative failure after needle-prick injury, observed by birefringence imaging (g), quantified in (h). This repair defect correlates with the marked proliferative defect (EdU, white) observed within the injury site after needlestick skeletal muscle injury [myosin heavy chain (MyHC), skeletal muscle visualization, magenta] (i). Growth of mutant larvae within the injury site was reduced to the constitutive levels observed outside the injury site, and the mutant failed to elicit the additional proliferative response required to sustain repair. was emphasized. Moreover, these observations recapitulate those seen after ablation of host and mmp9-expressing macrophages. Quantification (j). k to t, also using CRISPR/Cas9 to target exon 2 of ccr5 (utilizing two gRNAs) to induce a frameshift and subsequent premature stop codon (referred to as ccr5p.Pro24Leufs*28, ccr5 ^pc42 ) Deletion-insertion mutations were generated (k). (l) Schematic representation of the two exons of ccr5, the mutation site (513 bp deletion and 90 bp insertion), and the transcript of the mRNA. Primer binding sites for RT-PCR are also shown. (m) RT-PCR analysis of ccr5 cDNA revealed a 234 bp product corresponding to the mutant transcript (red arrow). A wild-type transcript corresponding to the 657 bp product (red arrow) is absent in the mutant. Both PCR fragments were sequence verified. The mutant transcript lacks most of the chemokine domain encoded by exon 2 of ccr5, so the mutant protein, if translated, will lack the ligand binding site and will be non-functional. Similar to the nampta mutants above, the ccr5 mutants show comparable macrophage dynamics (representative images, n and quantification, o) and macrophage-stem cell interactions (p, n=20 observations) to their wild-type siblings. ) is shown. In addition, this mutant reflects the phenotypic defect described above for the nampta mutant, due to defective muscle stem cell proliferation at the wound site [s, phalloidin-labeled muscle, quantification (t)]. Showed marked skeletal muscle repair defects (q), (r) upon injury. e, h, j, o, r, t, two-way ANOVA and Tukey's multiple comparison test.

Immune cell-specific gene editing in nampta and namptb in larval zebrafish. ad, Validation of macrophage-specific nampta gene editing strategies. (a) Macrophages were isolated by FACS at 3 dpf from mpeg1-Cas9 larvae injected with namptag RNA. DNA isolated from these cells was used to generate PCR amplicons of the region encompassing the gRNA target site. Sanger sequencing of this amplicon confirmed the presence of a sequence disruption starting a few bases upstream of the PAM site. (b) Nampt protein expression at the wound site was assessed in namptagRNA-injected mpeg1-Cas9 larvae after needlestick muscle injury (white dotted line). Gene-edited larvae showed a marked reduction in Nampt expression (magenta) within the injured area (n=12). c, Macrophages isolated by FACS from gene-edited larvae were assayed for Nampt functionality by measuring NAD ⁺ /NADH levels using a luminescence-based assay. Macrophages isolated from control untreated larvae were used to measure baseline NAD ⁺ /NADH levels in zebrafish larval macrophages. Larval macrophages treated with the Nampt enzyme inhibitor GMX1778 were used to identify NAD ⁺ /NADH levels present in macrophages in the absence of Nampt function. In addition, larval macrophages treated with NMN, the major product of Nampt's rate-limiting enzymatic reaction, were used to set the maximum threshold for assay sensitivity. It was found that macrophages isolated from mpeg1-Cas9 larvae injected with namptag RNA exhibited decreased NAD ⁺ /NADH levels, reflecting a loss of function of Nampt activity within macrophages present in these larvae. This assay can also detect residual enzymatic activity of Namptb that is not affected by this gene-specific targeting approach. Mean ± standard deviation. One-way ANOVA, multiple comparison test with Dunnett's post-hoc test. df. Post-wounding assay of macrophage (yellow) dynamics in nampta gene-edited larvae showed no observable changes from control larvae (d, n=10). Nampta gene-edited larval macrophages located within the injured area (dotted white line) transitioned to a resident state at 2 dpi (e, quantification, f). These resident macrophages come to interact with pax3a ⁺ muscle stem cells present in the wound (g, representative of n=20 observations). hj, muscle stem cell-specific ccr5 gene editing does not affect the dynamics of injury-responsive (dotted white lines demarcate the wound site) macrophages and transition to a resident phenotype at 2 dpi (h, quantification transformation, i). Moreover, these ccr5 gene-edited Pax7b ⁺ muscle stem cells (cyan) show phenotypically wild-type interactions with resident macrophages (yellow) located at the injury site (j, n=20 of observations). representative example). nk, In zebrafish, the role of Nampt in muscle regeneration is dominated by Nampta rather than Namptb. (k) Namptb expression by in situ hybridization of the wound site, lateral view showed constitutive expression from 1-3 dpi at the wound site. Mpeg1-Cas9 larvae injected with two gRNAs targeting nmptb (schematic, l) showed modest skeletal muscle regenerative capacity by birefringence imaging (m) after needlestick injury. Quantification (n). oq, Macrophages, but not neutrophils, are the major and functional source of Nampta in muscle regeneration. nampta was specifically knocked down in neutrophils (using the mpx-Cas9 lineage, schematic, o), another important innate immune cell type present in regeneration. Using this approach, no regeneration defects were observed with birefringent imaging after needle-prick muscle injury (p). Quantification (q). f, i, n, q, mean±s.d. Two-way ANOVA with Tukey's multiple comparison test. NAMPT supplementation after acute skeletal muscle injury in zebrafish larvae promotes proliferation specifically at the injury site. ac, NAMPT supplementation does not alter immune cell responses to injury. Larvae muscle-injured by needle stick (white dotted line) were immediately supplemented with human recombinant NAMPT ₍₁₎ [hrNAMPT ₍₁₎ ] (4 dpf/0 dpi) and their immune cell dynamics assayed at 2 dpi. The numbers of macrophages [Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), yellow] and neutrophils [Tg(mpx:eGFP)cyan] were comparable to untreated controls (a). Quantification (b). Mean ± standard deviation. Two-way ANOVA with Tukey's multiple comparison test. Furthermore, the autophagic process within the injured area was compared by LysoTracker (magenta) and found to be comparable between control and hrNAMPT ₍₁₎ -supplemented larvae (a). Quantification (c). Mean ± standard deviation. Unpaired t-test. d, NAMPT and combination drug supplementation in the Tg (mpeg1:GAL4FF/UAS:NfsB-mCherry) strain for effects on proliferation (EdU, white) at the wound site (dotted white line, skeletal muscle visualized by phalloidin staining, magenta). assayed later. Four individual examples are provided for each group, and these images are representative of the quantification shown in Figure 4m. NAMPT supplementation not only increases wound site proliferation in the control setting, but also rescues the proliferation defect that accompanies macrophage ablation. However, because NAMPT acts on the Ccr5 receptor to elicit its proliferative response, inhibition of this receptor by CVC treatment resulted in a proliferative defect that resists NAMPT-mediated rescue. Furthermore, the Ccr5 ligand, CCL8, while promoting proliferation at the wound site, also promoted proliferation outside the wound site, highlighting the low specificity that NAMPT can exert. e, Satellite cell proliferation in mouse muscle injury supplemented with NAMPT. A gating strategy for separating proliferating satellite cell populations is shown. CD45 ⁻ , CD11b ⁻ , Ly6G ⁻ , CD31 ⁻ , VCAM-1 ⁺ and PAX7 ⁺ cells are considered satellite cells. A further satellite cell that proliferates is Ki67 ⁺ . Quantification 4 days after treatment is shown in Figures 4q-r. fg, Angiogenesis after mouse VML injury treated with NAMPT. (f) Muscle injury was treated with NAMPT (0.5 μg) delivered in fibrin or fibrin-only control and tissues were harvested 6 days after treatment. Representative regenerated muscle cryosections stained with CD31 (endothelial cells, yellow) and laminin (magenta). (g) Quantification of CD31-positive area (n=6 mice/group). hi, Immune cell profiles after muscle injury in mice supplemented with NAMPT. (h) Gating strategy for analyzing wound immune cell subsets in muscle injury. Gating strategies for a panel of neutrophils, macrophages, and T cell subsets are shown. (i) Percentages were calculated by the total number of viable cells in the tissue harvested (n=5 mice on day 6, n=6 mice on day 8). Mean ± S.E.M. E. M. Two-way ANOVA, Bonferroni post-hoc test for pairwise comparisons. j, Schematic representation of the role of injury-responsive MΦs in regulating muscle stem cell proliferation. After acute muscle injury (1), macrophages and muscle stem cells migrate to the injury site (2). About half of these injury-responsive macrophages reside at the wound site during repair (3). A subset of mmp9 ⁺ /mpeg1 ⁺ resident macrophages begins to actively interact with pax3a ⁺ muscle stem cells present in the wound (4). These macrophages actively secrete Nampta that binds Ccr5 on muscle stem cells (4'), activating signaling cascades that lead to muscle stem cell proliferation (5).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "isolated cell" means a cell that has been removed from the organism in which it was originally found, or the progeny of such a cell. The cells may, for example, have been cultured in vitro in the presence of other cells. Also, the cell is destined for later introduction into a second organism, or is destined for reintroduction into the organism from which the cell (or progeny of the cell) was isolated. There is also

The terms "isolated cell population" and the like refer to cell populations that have been removed and separated from mixed or heterogeneous cell populations. In some embodiments, an isolated population is a substantially pure population of cells as compared to a heterogeneous population from which the cells have been isolated or enriched.

In one embodiment, the present application allows for a pharmaceutical or physiologically active regenerating composition comprising 1 or 2 or 3 or 4 or 5 of:
(i) comprising or encoding a CCR5 interacting agent as defined herein (ii) tissue stem cells (e.g. satellite cells) or their progenitor cells or their progeny cells (iii) macrophages or their progenitor cells or their progeny cells (iv) a scaffolding or retention material; (v) a tissue delivery enhancing component;

In one embodiment, the application provides a cell composition comprising one or more of the stem cells, stromal cells, pre-satellite or satellite cells, pre-macrophages or macrophages or macrophage-derived factors described herein. In one embodiment, pluripotent "tissue stem cells" include pre-muscle cells or any pre-macrophage cells, which may be produced in an essentially native form, may be treated with heterologous factors or It may be modified to express self-factors. Similarly, the term pluripotent "tissue stem cell" can include the activated progeny of tissue stem cells.

Tissue stem cells, including muscle stem cells, can be isolated or derived (for ex vivo or in vitro or in vivo treatment).

Stem cells can be contacted with a medium or composition containing a CCR5 agonist for any length of time. For example, stem cells can be contacted with a CCR5 agonist for 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week or more. Stem cells can be induced or stimulated to differentiate into cell lineages selected from the group consisting of mesoderm, endoderm, ectoderm, neural, mesenchymal, and hematopoietic.

In some embodiments, the stem cells are human stem cells, multipotent adult stem cells, multipotent adult stem cells, or embryonic stem cells.

Adult human stem cells undergo mitosis, usually leaving one of the daughter cells as the stem cell. Adult tissues are composed of one or more resident progenitor or stem cells that occupy specific niches within the tissue and actively sense and respond to its local environment. Each tissue usually has its own resident stem cells that promise to produce progeny that differentiate into a specific range of cell types. Muscle tissue is composed of satellite cells committed to producing myogenic cells. Stem cells of this kind include mesenchymal stem cells (MSC), which produce many different types of cells such as muscle, cartilage, bone, and fat, hematopoietic stem cells (HSC), which produce all blood cells and the hematopoietic system, and neural stem cells (NSC). have also been extensively studied. All tissues have resident stem cell populations, including heart, intestine, and liver. Adult stem cells are usually multipotent, which refers to cells that can differentiate into some, but not all, cells from all three germ cell layers. A multipotent cell is therefore a partially differentiated cell. For example, MSCs can be obtained by many methods well known in the art. See U.S. Patent Nos. 5,486,358, 6,387,367, and 7,592,174, and U.S. Patent Application Publication No. 2003/0211602. MSCs may be derived from bone, fat, and other tissues in which they reside. "Origin" does not mean direct origin, but merely indicates the original place of origin.

In one embodiment, the stem cells are non-embryonic stem cells or adult multipotent stem cells.

In one embodiment, the stem cells are HSCs or MSCs.

Adult stem cells expressing CCR5 can be stimulated to undergo differentiation by exposure to CCR5 interacting agents described herein. Cells are monitored for altered expression of, for example, myogenic regulatory factors known in the art.

Cells can be cultured in standard media or in specifically designed media.

Expression in cells can be modified by techniques well known in the art.

Induced pluripotent stem cells and partially induced pluripotent stem cells are convenient sources of stem cells. They can be derived from differentiated adult cells such as human foreskin cells.

Human iPS cells can be produced by introducing specific reprogramming factors into non-pluripotent cells. Reprogramming factors include, for example, Oct3/4, Sox family transcription factors (e.g., Sox1, Sox2, Sox3, Sox15), Myc family transcription factors (eg c-Myc, 1-Myc, n-Myc), Kruppel-like family (KLF) transcription factors (eg KLF1, KLF2, KLF4, KLF5) and/or NANOG , LIN28 and/or related transcription factors such as Glis1. For example, reprogramming factors can be introduced into cells using one or more plasmids, lentiviral vectors, or retroviral vectors. In some cases, the vector can be integrated into the genome and removed after reprogramming is complete. In some embodiments, the vector is non-integrating [e.g., positive-stranded single-stranded RNA species derived from non-infectious (non-packaging) self-replicating Venezuelan equine encephalitis (VEE) virus, Simplicon RNA Reprogramming Kit, Millipore, SCR549 and based on SCR550]. The Simplicon RNA replicon is a synthetic intratranscribed RNA that expresses all four reprogramming factors (OKG-iG, Oct4, Klf4, Sox2, and Glis1) in polycistronic transcripts and is capable of self-renewal with a limited number of cell divisions. It is possible. Human induced pluripotent stem cells made with the Simplicon Kit are called "integration-free" and "footprint-free." Human iPS cells can also be generated using miRNAs, small molecules that mimic the action of transcription factors, lineage specifiers, and the like. Human iPS cells are characterized by their ability to differentiate into any cell of the three vertebrate germ layers: endoderm, ectoderm, and mesoderm. Human iPS cells are also characterized by unlimited proliferation under appropriate in vitro culture conditions. Human iPS cells express alkaline phosphatase, SOX-2, OCT-4, Nanog and Tra-1-60 markers.

The terms "naive" and "primed" distinguish different pluripotent states of human iPS cells. The properties of naïve and primed iPS cells have been described in the art literature. Naive human iPS cells exhibit a pluripotent state similar to ES cells of the inner cell mass of preimplantation embryos. Such naive cells have not been primed for lineage specification and commitment. Female naive iPS cells are characterized by having two active X chromosomes. In culture, self-renewal of naive human iPS cells is dependent on leukemia inhibitory factor (LIF) and other inhibitors. Cultured naive human iPS cells exhibit a clonal morphology characterized by rounded, domed colonies and lack of apical-basal axial polarity. Cultured naive cells can additionally display one or more pluripotency markers, as described elsewhere herein. The doubling time of naive human iPS cells in culture can be 16-24 hours under appropriate conditions.

Primed human iPS cells exhibit a pluripotent state similar to post-implantation epiblast cells. Such cells are primed for lineage specification and commitment. Female primed iPS cells are characterized by having one active and one inactive X chromosome. In culture, self-renewal of primed human iPSCs is dependent on factors such as fibroblast growth factor (FGF) and activin. Cultured primed human iPSCs exhibit a clonal morphology characterized by an epithelial monolayer and exhibit apical-basal axial polarity. Under appropriate conditions, the doubling time in culture of primed human iPSCs can be 24 hours or more, depending on the levels from the adult cells from which they are derived.

Embryonic stem cells (ESCs) are characterized by pluripotency, with the ability to differentiate into cell types characteristic of all three germ cell layers (endoderm, mesoderm and ectoderm) under different conditions. Pluripotent cells are primarily characterized by their ability to differentiate into all three germ cell layers. In some embodiments, pluripotent cells are undifferentiated cells. Pluripotent cells also have the ability to divide in vitro for over a year or over 30 passages.

ESCs are pluripotent stem cells, typically of the inner cell mass of the blastocyst (see US Pat. Nos. 5,843,780, 6,200,806). Such cells can also be obtained from the inner cell mass of blastocysts obtained from somatic cell nuclear transfer (U.S. Pat. Nos. 5,945,577; 5,994,619; 6,235; 970). Properties of exemplary differentiable embryonic stem cells include, but are not limited to, gene expression profile, proliferation potential, differentiation potential, karyotype, responsiveness to particular culture conditions, and the like.

In one embodiment, the stem cells are adult.

In one embodiment, the stem cells are autologous or xenogeneic to the subject.

In one embodiment, the stem cells are mammalian or human.

Stem cells, including macrophages and satellite cells, can be prepared by art-recognized methods and as described herein, including the use of iPSCs and optionally gene editing procedures.

In one embodiment, isolated macrophages or stem cell-derived macrophages are engineered to express a truncated NAMPT peptide as described herein. Generally, M2-type macrophages are selected or provided.

In one embodiment, stem cells are contacted in vitro, ex vivo or in vivo with a CCR5 interacting agent to induce activation and proliferation as described herein. In vitro or ex vivo treated stem cells may be introduced into a wound site to effect repair, or may be administered systemically to effect regeneration of damaged tissue, as described herein. and may be administered systemically to treat or ameliorate muscle-related conditions.

A CCR5 agent or cells expressing it can be administered in the form of a functionalized hydrogel, alone or together with cells for transplantation. Such hydrogels or similar biomaterials or scaffolds improve the efficiency of implantation at the wound site.

Hydrogels may be ECM-based, such as fibrin-based. Alternatively, hydrogels may be non-ECM-based, such as acrylamide-based using RAFT technology (Chiefari et al Macromol. 31:5559-5526, 1998 and Fairbanks et al Advanced Drug Delivery Reviews 91: 141-152, 2015 ). Suitable materials have the desired mechanical and physical properties to modulate release kinetics and for tissue regeneration, as is known in the art.

In one embodiment, satellite cells are encapsulated in a CCR5 functionalized hydrogel or other biomaterial.

Kits containing the cell compositions and/or agents described herein are also provided. Kits suitable for muscle repair or regeneration are specifically contemplated. The CCR5 agonist can be pre-formulated for administration or the ingredients for formulation can be provided with the kit. CCR5 agonists, for example, are formulated in hydrogels or other support vehicles for topical application. The CCR5 agonist can be, for example, lyophilized or liquid.

The terms "protein", "polypeptide" and "peptide", used interchangeably herein, refer to coded and non-coded amino acids, as well as chemically or biochemically modified or derivatized amino acids. Included are polymeric forms of amino acids of any length, including The term also includes modified polymers, such as polypeptides with modified peptide backbones.

A protein is one that has an "N-terminus" and a "C-terminus." The term "N-terminus" refers to the starting point of a protein or polypeptide, terminated by an amino acid with a free amine group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (protein or polypeptide), which in nature is terminated by a free carboxyl group (-COOH). In this application, references to C-terminal and N-terminal fragments broadly describe the region of the full-length molecule from which the selected portion is derived, but excludes the full-length or native molecule. C-terminal fragments need not, but may include, all C-terminal amino acids, and N-terminal fragments need not, but may include, all N-terminal amino acids.

The present application discloses and enables the use of various CCR5 interacting agents based on the findings first described in the Examples. Specifically, peptide CCR5 interacting agents are provided in the form of NAMPT and functional C-terminal fragments thereof.

Many peptide modifications are known in the art and are included to stabilize peptides against serum proteases or to facilitate intracellular localization. Some such modified peptides can be expressed from nucleic acids within the cell, others are produced synthetically. Similarly, if it is desired to target a CCR5 interacting agent to one or more specific cell types, it can be ex vivo manipulation of target cells, or specific binding to target cells or tissues such as ECM binding sites. Incorporation of targeting sites can be accomplished by any of the methods known in the art.

A "conservative amino acid substitution" is one in which a naturally occurring or non-naturally occurring amino acid residue is replaced with a naturally occurring or non-naturally occurring amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include basic side chains (e.g. Lys, Arg, His), acidic side chains (e.g. Asp, Glu), uncharged polar side chains (e.g. Gly, Asn, Gln, Ser, Thr, Tyr , Cys), nonpolar side chains (e.g. Ala, Val, Leu, Ile, Pro, Phe, Met, Trp), β-branched side chains (e.g. Thr, Val, Ile) and aromatic side chains (e.g. Phe , Trp, His). Thus, for example, a predicted nonessential amino acid residue in CCR5 may be replaced with another amino acid residue from the same side chain family. Other examples of permissible substitutions may be based on isosteric considerations (eg norleucine for methionine) or other properties (eg 2-thienylalanine for phenylalanine). A complete subgroup of amino acids is shown in Table 2 and exemplary substitutions are shown in Table 3.

CCR5-interacting peptides may contain modifications known to modify the pharmacokinetic characteristics of the peptide, such as increasing protease resistance in vivo.

In one embodiment, the peptide comprises a linker or spacer such as GGS or repeats of GGS and variants known in the art), modified or unnatural or unprotected amino acids, modified side chains, modified backbone, terminal modification groups, or contains one or more modified spatial constraints, or is a D-retro-inverso peptide. In one embodiment, the peptide is a pseudopeptide, peptoid, azapeptide, cyclized, stapled, ether or lactam peptide, or contains spatial constraints.

In one embodiment, the CCR5-interacting agent or peptide is optionally conjugated with a lipid, carbohydrate, polymer, protein, nanoparticle, peptide, proteoglycan, antibody or fragment or antigen-binding form thereof, aptamer, or nucleic acid, or other Attached/bonded/expressed in a manner.

In one embodiment, the CCR5-binding agent specifically binds to muscle cells or muscle cell tissue or related structures, such as ECM.

In one embodiment, CCR5 interacting agents include physiologically or pharmaceutically acceptable salts, hydrates, stereoisomers, and prodrugs.

In one embodiment, non-essential amino acids may be altered. References to "non-essential" amino acid residues refer to residues that can be altered from the wild-type sequence of a polypeptide (e.g., secNAMPT) without losing or substantially altering its ability to bind endogenous or heterologous CCR5. means the base.

In one embodiment, the CCR5 interacting agent is an amino acid sequence set forth in one of SEQ ID NOs: 1-4, or at least 70%, 75%, 80%, 85%, 86%, 87%, 88% thereof %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences, or It may be encoded with one or more tissue delivery-enhancing or signaling-enhancing moieties. In one embodiment, a CCR5 interacting agent with a small number of substituted, added or deleted residues is a cif motif identified herein (eg, between 40 and 40 within the C-terminal portion of NAMPT that mediates CCR5 binding). 100 residues) and retains the ability to interact with and activate satellite cells.

In one embodiment, the CCR5 interacting agent has 1, 2, 3, 4, 5 or 6 conservative or non-conservative amino acid substitutions, deletions or additions to the above sequence but has CCR5 interacting activity contains or encodes an amino acid sequence that retains

In one embodiment, a CCR5 interacting agent comprises a nucleic acid molecule capable of expressing a CCR5 interacting peptide.

Examples of suitable polynucleotide sequences are those encoding peptide/polypeptide sequences set forth in SEQ ID NOs:6-9. In certain embodiments, the disclosure provides a polynucleotide comprising a nucleic acid sequence comprising a coding sequence for an encodeable CCR5 peptide or an encodeable CCR5 interacting agent. For example, in some embodiments, the nucleic acids of the present disclosure are any one of SEQ ID NOs: 6-10 and at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% comprising, consisting essentially of, or from Become.

In one embodiment, the nucleic acid molecule is RNA or DNA or RNA:DNA or chemically modified forms thereof.

In one embodiment, a portion of at least one nucleotide (eg, cytidine and/or uracil) is chemically modified to enhance in vivo stability.

In one embodiment, the nucleic acid is in the form of a viral or non-viral vector.

In one embodiment, the CCR5 interacting agent is administered to cells ex vivo. The invention encompasses the use of genetically engineered cell depots (eg, CAR T-cells, TCRs, genetically engineered macrophages, etc.).

In one embodiment, the CCR5-interacting agent comprises an antibody or antibody fragment that specifically targets the agent to target cells such as muscle stem cells.

In one embodiment, the application provides a pharmaceutical or physiological composition comprising a CCR5 interacting agent as defined herein above.

The present application provides a method of treating muscle damage or treating a person with a reduced or suboptimal ability to repair or regenerate muscle, wherein CCR5 is sufficient to stimulate muscle stem cell proliferation and muscle regeneration. It enables a method comprising administering to a subject an effective amount of a composition comprising an interacting agent.

In another aspect, the disclosure relates to agents or compositions comprising the CCR5-interacting agents described herein.

Compositions include vehicles that are biologically or otherwise objectionable, physiologically or pharmaceutically acceptable. Pharmaceutically acceptable salts, esters, prodrugs, or derivatives of the compounds described herein refer to salts, esters, prodrugs, or derivatives that are biologically or otherwise undesirable. .

In some embodiments, the agent is modified. Peptide and drug activity are tolerant of additional sites, flanking residues and substitutions within defined boundaries. Similarly, backbone modifications and substitutions, side chain modifications, and N- and C-terminal modifications are conventional in the art. Generally, modifications are to enhance stability or pharmacological profile, targeting/delivery. For example, cyclization or stapling of peptides is conventionally performed to increase peptide stability. In another embodiment, the peptide or drug is in the form of micro- or nanoparticles or foams, gels, liposomes, conjugates or fusion proteins containing moieties adapted for stability, target tissue delivery or specificity is.

In one embodiment, agents or nucleic acids encoding them are assembled in liposomes, hydrogels, emulsions, viral vectors, virus-like particles or virosomes, as appropriate.

In one embodiment, specific binding sites such as antibodies or antibody fragments or mimetics are used to target agents to the muscle environment.

In one embodiment, the peptide agent is delivered via delivery of mRNA, gene editing such as by CRISPR components, or biosynthesis in vivo such as in bacteria or cells.

Compositions generally include a CCR5-interacting peptide, peptidomimetic, or encoding nucleic acid, as appropriate, and a pharmaceutically acceptable carrier and/or diluent. In one embodiment, the carrier may be a nanocarrier.

In one embodiment, a CCR5 interacting agent of the present disclosure is not a naturally occurring molecule, but instead is a modified form of a naturally occurring molecule that does not possess the specific features or functions of the naturally occurring full-length molecule. is. For example, it may not have NAMPT enzymatic activity.

In another embodiment, the CCR5-interacting peptide is constrained by a linker covalently joining at least two amino acids in the peptide. Various cyclization strategies are known in the art to enhance stability and cell permeability.

In some embodiments, the CCR5 interacting agent is delivered in the form of a vector comprising a nucleic acid molecule encoding it, or a prodrug thereof, or a nucleic acid molecule encoding it, or a prodrug thereof. . In one embodiment, the nucleic acid is mRNA. CCR5-interacting agents may bind to the surface of muscle stem cells or function internally to stimulate signaling and proliferation.

In another embodiment, the CCR5-interacting agent is combined with amphipathic agents such as lipids, in addition to other pharmaceutically acceptable carriers, to form micelles, insoluble monolayers, liquid crystals or lamellar layers in aqueous solutions. exist in association as

In one embodiment, the present disclosure provides compositions comprising a CCR5-interacting agent described herein that interacts with endogenous CCR5 protein for use as a pharmaceutical or for use in therapy. enable.

In another aspect, the present disclosure enables the provision of compositions for stimulating proliferation of muscle stem cells comprising a CCR5-interacting peptide or a nucleic acid molecule capable of expressing said peptide.

In one embodiment, the subject composition is co-administered with a second physiologically active, therapeutic or prophylactic or regenerative agent. Exemplary cytokines include, but are not limited to, one or more of IGF-1, TGF-b, GDF-5, bFGF, PDGF-b3, IL-4.

In another aspect, the disclosure provides the use of CCR5 interacting agents in the manufacture of a medicament for stimulating muscle regeneration or in stem cell therapy.

In one embodiment, the present application provides screening assays for CCR5-interacting agents as described herein, comprising assessing the agent's ability or indication thereof to induce muscle stem cell proliferation and muscle generation. is.

Peptide-based therapeutics are useful molecules because they are known to be potent and selective against biological targets that are difficult to manipulate with small molecules. Modified peptides have been developed to improve the pharmacokinetic properties of linear peptides.

Peptides of the present disclosure comprise amino acids. Reference to "amino acid" includes naturally occurring or non-naturally occurring amino acids.

Peptide compounds can be modified routinely and routinely by the addition of sites, flanking peptide residues, and substitutions within the parameters recognized. Peptides can further comprise modified backbones, side chains, peptide bond replacements, and routine terminal modifications using standard peptide chemistry.

Amino acids incorporated into the amino acid sequences described herein may be L-amino acids, D-amino acids, L-β-homoamino acids, D-β-homoamino acids or N-methylated amino acids, sugar amino acids, and/or It may be a mixture. Unnatural amino acids may not be recognized by proteases and thus may alter half-life. In one embodiment, a D-retroinversion sequence is employed.

Unnatural amino acids include chemical analogues of the corresponding naturally occurring amino acids. Examples of unnatural amino acids and derivatives include 4-aminobutyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t -butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienylalanine and/or the D-isomer of an amino acid.

In one embodiment, the peptide is modified to enhance its pharmaceutical properties using art-recognized modifications. Peptides may be substituted, eg, alanine-substituted, or may be substituted with crosslinkable sites, and/or may be linked. Suitable residues may contain additional α-carbon substitutions selected from hetero-lower alkyl, heteromethyl, ethyl, propyl and butyl. Peptide bond replacements such as trifluoroethylamine are used to produce more stable and active peptidomimetics.

Thus, cyclic or stapled peptides, peptoids, peptomers, and peptidomimetic forms of peptides are included.

Peptidomimetics and cyclic peptides are protected from exopeptidases. After cleavage of the peptide, it can be circularly ligated from the N-terminus to the C-terminus. This can be done by direct coupling or by introducing specific functional groups and cyclization through a binary reaction. Examples include side chain modifications such as linkers to form Cys-Cys disulfide bridges, macrolactam peptides, thioether peptides, staple peptides, and the like. Click variants are particularly effective for cyclizing peptides. Another approach is to couple 2-amino-d,1-dodecanoic acid (Laa) to the N-terminus and replace Asn with lipoamine.

A more rigid structure can be obtained by using a more rigid backbone with heterocycles, N-methylated amine linkages, and methylated α-carbon atoms.

Among the techniques used for stapling peptides, the two-component double-copper-catalyzed azide-alkyne cycloaddition (CuAAC) strategy confines peptides to a bioactive conformation while improving pharmacokinetic properties. . In addition, this strategy uses synthetically facile, non-natural azide amino acids to facilitate functionalization of staples, fluorescently labeled tags, and photoswitchable linkers. Independent functionalization of the staples is believed to be particularly useful as multiple functionalities are added to the staple rather than the N- or C-terminus of the peptide. Moreover, this approach can generate different functionalized staple peptides with a single linear peptide, facilitating the exploration of different functionalities on the linker and thus the properties of the whole peptide.

Azapeptides are peptide analogues in which one or more amino acid residues have been replaced with semicarbazide. Such substitution of nitrogen at the center of the α-carbon creates a conformational constraint in which the peptide bends from a linear shape around the aza-amino acid residue. The turn conformation of the resulting azapeptide is observed by X-ray crystallography and spectroscopy, and also predicted based on computational models. In biologically active peptide analogues, aza-substitutions have increased activity and selectivity, as well as improved properties such as prolonged duration of action and metabolic stability.

Half-life can also be increased by terminal acylation or amidation. Peptoids are produced with N-alkylated oligoglycine side chains. In some embodiments, peptides may be acetylated, acylated (e.g., lipopeptides), formylated, amidated, phosphorylated (on Ser, Thr and/or Tyr), sulfated or glycosylated. good.

As used herein, the term "macrocyclization reagent" or "macrocyclization-forming reagent" is used to prepare peptidomimetic macrocycles by mediating a reaction between two reactive groups. refers to any reagent obtained. Reactive groups can be, for example, azides and alkynes, where macrocyclization reagents include, but are not limited to, reagents that provide reactive Cu(I) species such as CuBr, CuI or CuOTf, and Cu Cu(II) salts such as (CO2CH3)2, CuSO4, and CuCl2, which can be converted in situ to active Cu(I) reagents by the addition of reducing agents such as ascorbic acid or sodium ascorbate. Cu reagents such as Cu(II) salts are included. Macrocyclization reagents are Ru reagents known in the art, such as, for example, Cp*RuCl(PPh3)2, [Cp*RuCl], or other Ru reagents that can provide reactive Ru(II) species. may additionally be included. In other embodiments, the reactive group is a terminal olefin. In such embodiments, the macrocyclization or macrocyclization reagent is a metathesis catalyst including, but not limited to, stabilized late transition metal carbene complex catalysts such as Group VIII transition metal carbene catalysts. For example, such catalysts are Ru and Os metal centers with a +2 oxidation state, an electron count of 16, and pentacoordination. Additional catalysts are described in Grubbs et al., "Ring Closing Metathesis and Related Processes in Organic Synthesis" Acc. Chem. Res. 1995, 28,446-452, and US Pat. No. 5,811,515. In yet another aspect, the reactive group is a thiol group. In such embodiments, the macrocyclization reagent is a linker functionalized with two thiol-reactive groups, such as, for example, halogen groups.

In one embodiment, the peptidomimetic macrocycle has improved structural stability, increased affinity for a target, increased resistance to proteolysis, etc., compared to a corresponding non-macrocyclic polypeptide. Exhibits biological properties. In another embodiment, the peptidomimetic macrocycle comprises one or more alpha helices and/or exhibits an increased degree of alpha helicity in aqueous solution compared to a corresponding non-macrocyclic polypeptide.

For example, it is possible to analyze the sequence of a peptide and substitute the azide- and alkyne-containing amino acid analogs of the invention at appropriate positions. Appropriate positions are selected for which molecular surface(s) of secondary structure are required for biological activity, and thus the macrocyclization linker of the present invention sterically aligns the surface(s) required for biological activity. This is determined by confirming that macrocyclic structures can be formed across other surfaces without blocking. Such determinations may be made by methods such as X-ray crystallography of complexes of secondary structure and natural binding partners to visualize residues (and surfaces) important for activity ( and surface), by sequential mutagenesis of residues in the secondary structure, or by other methods. Upon such determination, appropriate amino acids are substituted with the amino acid analogs and macrocycle-forming linkers of the invention. For example, in the case of a helical secondary structure, one surface of the helix (eg, the molecular surface extending longitudinally along the axis of the helix and radially 45-135 degrees with respect to the axis of the helix) is critical for biological activity. It may be necessary to contact another biomolecule in vivo or in vitro. In such cases, the macrocycle-forming linker is designed to connect two carbons of the helix while extending longitudinally along the surface of the helix in portions not directly required for activity.

The peptidomimetic macrocycle may be helical in aqueous solution. For example, a peptidomimetic macrocycle may exhibit increased helical conformation in aqueous solution compared to a corresponding non-macrocyclic polypeptide. In some embodiments, peptidomimetic macrocycles exhibit increased thermal stability compared to corresponding non-macrocyclic polypeptides. In other embodiments, peptidomimetic macrocycles exhibit enhanced biological activity compared to corresponding non-macrocyclic polypeptides. In still other embodiments, peptidomimetic macrocycles exhibit increased resistance to proteolysis compared to corresponding non-macrocyclic polypeptides. In still other embodiments, the peptidomimetic macrocycle exhibits an enhanced ability to penetrate living cells compared to the corresponding non-macrocyclic polypeptide.

The term "amino acid analog" refers to molecules that are structurally similar to naturally occurring amino acids and that can substitute for the amino acids in forming peptidomimetic macrocycles. Amino acid analogs include, but are not limited to, compounds containing one or more additional methylene groups between the amino and carboxyl groups, or other than replacing the amino or carboxyl groups with similar reactive groups (e.g., Substitution of a primary amine with a secondary or tertiary amine, or substitution of a carboxy group with an ester) includes compounds that are structurally similar to the amino acids defined herein.

The peptide may contain an N-terminal acetyl, formyl, myristoyl, palmitoyl, carboxyl, 2-furosyl, or C-terminal hydroxyl, amide, ester or thioester group. In one embodiment, the peptide is acetylated at the N-terminus and amidated at the C-terminus. In one embodiment, a chelating agent such as DOTA, DPTA is introduced. Peptides can be modified by, for example, pegylation, lipidation, XTENylation, pasylation, and other approaches that extend the half-life of the peptide in vivo or in vitro. In one embodiment, pegylation is used to increase peptide solubility and bioavailability. Various forms of pegs are known in the art, including HiPegs, branched and forked Pegs, releasable Pegs, heterofunctional Pegs with terminal group NHS esters, maleimides, vinyl sulfones, pyridyl disulfides, amines and carboxylic acids. mentioned. Examples of therapeutic pegylated peptides include pegfilagrastin (Neulasta) from Amgen.

A linker or spacer may be an amino acid or nucleic acid, or other atomic structure known in the art, and is typically 2-10 amino acids or nucleotides in length. The spacer is sufficiently flexible to allow correct orientation of the CCR5-interacting constructs described herein, such as nanoparticles, antibody fragments, liposomes, those containing cell-penetrating and/or intracellular delivery moieties. should. One form of spacer is an IgG-derived hinge region, suitable for use when the construct contains an antigen-binding portion for cell targeting.

Antigen-binding molecules include, for example, extracellular receptors, antibodies or antibody fragments (including molecules such as ScFv), and the like. A signal peptide may be present at the N-terminus. Bispecific antibodies capable of selectively binding to more than one epitope are known in the art and can be used to bind to the CCR5 interacting agents of the invention, for example to bind to the muscle environment or other substrates. can be used.

In one embodiment, the peptide is conjugated or otherwise attached (covalently or non-covalently) to a delivery agent. In one embodiment, the delivery agent delivers the peptide to a tissue, target cell or cell population.

Derivatives of CCR5 interacting agents include biologically active fragments thereof, including the structures or orthologs described herein. Systematic truncation or alanine scanning or modeling around conserved motifs can be routinely performed to identify minimal peptides with CCR5 agonistic effects.

Derivatives also include molecules that have a predetermined percent amino acid or polynucleotide sequence identity over a comparison window after optimal alignment. In one embodiment, the percent identity is at least 80%-99%, including any number between 80-99.

Suitable assays for peptide or drug biological activity are known to those of skill in the art and are described herein.

In some embodiments, markers of peptide activity include upregulation of satellite cell signaling (eg, MAPK), stem cell and myoblast proliferation and differentiation.

In one embodiment, the CCR5-interacting agent is modified at non-naturally occurring amino acid residues. The portion consists of detectable labels, non-naturally occurring amino acids as described herein, reactive groups, fatty acids, cholesterol, lipids, bioactive carbohydrates, nanoparticles, small molecule drugs, and polynucleotides. can be selected from the group. In certain embodiments, this moiety is a detectable tag label. In one example, the detectable label is selected from the group consisting of fluorophores, fluorogenic substrates, luminogenic substrates, and biotin. Art-recognized tags or labels include affinity agents and moieties for detection include fluorescent and luminescent compounds, metals, dyes. Other useful moieties include affinity tags, biotin, lectins, chelators, lanthanides, fluorochromes, FRET acceptors/donors.

In one embodiment, the CCR5-interacting agent, which may include a detectable label, accompanies in the kit a modified control version of the agent in which conserved residues of the CCR5 agent are replaced with, for example, alanine. Kits containing these agents are commercially available and can be used for screening or therapeutic purposes.

Peptides of this type are described, for example, in Sambrook et al., MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbour Press, 1989), especially Chapter 16 and 17, Ausubel, Current Protocols IN MOLECULAR BIOLOGY & Sons, Inc. 1994-1998), especially Chapter 10 and 16, and Curgan, Current It can be obtained by applying recombinant nucleic acid technology as described in PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), especially chapters 1, 5 and 6.

Alternatively, peptides of this type can be synthesized using conventional liquid-phase synthesis, or the recently developed technique of solid-phase synthesis. For example, as described by Atherton and Sheppard in SOLID PHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press at Oxford University, Oxford, England, 1989, see especially chapter 9), or by Roberge et al. (1995 Science 269:202). In-solution or solid-phase synthesis is mentioned first.

So far, the synthesis of azapeptides has been hampered by cumbersome solution-phase synthetic routes for selective addition of hydrazine functional groups. Recently, submonomer synthetic methods have enabled the addition of diverse side chains onto a common semicarbazone intermediate, providing the means to construct azapeptide libraries by solution and solid-phase chemistry. The submonomer strategy introduces semicarbazone, deprotonation, N-alkylation, orthogonal deprotection to introduce aza residues into the peptide chain. The desired azapeptide is obtained by aminoacylating and elongating the resulting semicarbazide. In addition, many chemical transformations have been carried out using orthogonal chemical reactions (Michael addition, N-arylation, etc.) of semicarbazone residues. Also, oxidation of azaglycine residues has yielded azopeptides that react in alicyclic reactions (such as Diels-Alder and Alder-ene chemistry). Most of these transformations of azaglycine residues were developed by the Lubell laboratory, and such chemistries have found application in the synthesis of ligands with biological activity that show promise in the treatment of diseases such as cancer and age-related macular degeneration. It has been. Azapeptide analogues of growth hormone-releasing peptide-6 (His-d-Trp-Ala-Trp-d-Phe-Lys-NH2, GHRP-6), for example, are explored as ligands for cluster of differentiation 36 (CD36) receptors. It has shown promising potential for the development of therapeutic agents for angiogenesis-related diseases such as age-related macular degeneration and arteriosclerosis. Azapeptides have also been used to generate structurally constrained second mitochondrial-derived caspase activator (Smac) mimetics that show promising apoptosis-inducing activity in cancer cells. Anticancer agents cilengitide, cyclo[RGDf-N(Me)V], and their parent compounds, which show efficacy against metastasis of human tumors and angiogenesis caused by tumors by performing azascan using the synthesis of cyclic azapeptide derivatives cyclo(RGDfV) conformation-activity correlation has been studied. An invention relating to the synthesis and application of azapeptides is described in Acc Chem Res. 2017 Jul 18; 50 (7): 1541-1556.

Alternatively, peptides can be produced by digesting the adapter polypeptide with proteases such as EndoLys-C, EndoArg-C, EndoGlu-C and Staphylococcus V8-protease. Digested fragments can be purified, for example, by high performance liquid chromatography (HPLC) techniques. A possible approach to optimizing the pharmacodynamic parameters of peptides and peptide analogues is Werle M. et al (2006) Strategies to improve plasma half-life time of peptide and protein drugs amino Acids 30(4):351- 367, and Di L (2014) Strategic approaches to optimizing peptide ADME properties AAPS J 1-10.

CCR5-interacting peptides can be stabilized, eg, via nanoparticles, liposomes, micelles, or as known in the art, eg, PEG. Methods of forming liposomes are described in Prescott, Ed. Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p.33 et seq., the contents of which are incorporated herein by reference. It is Polymer nanoparticles ideally use surfactants that are non-toxic and not physically adsorbed to the nanoparticles. In one aspect, biodegradable surfactants are used. For example, biodegradable, poly(ethylene glycol) (PEG)ylated N-(2-hydroxypropyl)methacrylamide (HPMA)-based surfmers are synthesized and used to stabilize lipophilic NPs. In particular, the NP core is formed from macromonomers consisting of polylactic acid (PLA) chains functionalized with HPMA double bonds. The nanoparticle-forming polymer chains are composed of a biocompatible, water-soluble homogeneous poly(HPMA) backbone and hydrolyzable PEG and PLA pendants that ensure complete degradability of the polymer. The stability of synthesized surfactants has been investigated in both emulsion-free and solution-free radical polymerizations followed by flash nanoprecipitation of the resulting amphiphilic copolymers.

Other stabilizing or heterologous moieties include NMEG, ECM binding, syndecan binding albumin, albumin binding proteins, immunoglobulin Fc domains, and the like.

Conventional Fc-fusion proteins and antibodies are examples of non-induced interaction pairs, but various antibody-engineered Fc domains are described by Spiess et al. (2015) Molecular Immunology 67 (2A): 95-106 is designed as an asymmetric interaction pair. An Fc conjugate can comprise an amino acid sequence derived from the Fc domain of an IgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgA1 or IgA2), IgE, or IgM immunoglobulin. Such immunoglobulin domains may contain one or more amino acid modifications (eg, deletions, additions, and/or substitutions) that promote hetero- or homo-dimeric or multimeric amyloid formation within a host cell.

In some embodiments, nanoparticles comprising CCR5-interacting agents can be further modified by conjugation of tissue-type specific binding agents, antibodies or fragments thereof known in the art.

Other suitable binding agents are known in the art and include antigen binding constructs such as affimers, aptamers or suitable ligands (receptors) or portions thereof.

Antibodies, such as monoclonal antibodies, or derivatives or analogs thereof, include, but are not limited to: Fv fragments, single-chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2 fragments, humanized Antibodies and antibody fragments, camelized antibodies and antibody fragments, and multivalent versions of the above. Multivalent binding reagents can also be used as appropriate, including but not limited to monospecific or bispecific antibodies such as disulfide stabilized Fv fragments, scFv tandem (scFv) fragments, diabodies, tribodies or ScFv fragments that are tetrabodies, typically in covalent or other stabilized (ie leucine zipper or helix stabilized) form.

As used herein, the term "antibody fragment" includes any portion of an antibody that retains the ability to bind to the epitope recognized by the full-length antibody. Examples of antibody fragments include Fab, Fab' and F(ab') ₂ , Fd, single-chain Fv (scFv), disulfide-bonded Fv (dsFv), and fragments comprising either the VL or VH region. , but not limited to them. An antigen-binding fragment of an antibody can consist of the variable region(s) alone, or part of the hinge region, CH1, CH2, CH3, or combinations thereof. Preferably, the antibody fragment contains all six CDRs of the whole antibody, although fragments containing fewer than all six CDRs may also be functional.

A “single-chain FV” (scFv) is an antigen-binding fragment in which the heavy chain variable region (VH) and light chain variable region (VL) of an antibody are linked by a single polypeptide. missing part or all. The binding between the VH and VL is made by short flexible peptides chosen to ensure proper three-dimensional folding of the VL and VH regions, while maintaining the binding specificity of the target molecule of the whole antibody from which the scFv was derived. ing. scFv lack some or all of the constant domains of antibodies.

Methods for making receptor-specific binding agents are generally known in the art, including antibodies and their derivatives as well as analogs and aptamers, especially based on natural ligands. Polyclonal antibodies can be produced by immunization of animals. Monoclonal antibodies can be prepared according to standard (hybridoma) techniques. Antibody derivatives and analogs, including humanized antibodies, can be prepared recombinantly by isolating DNA fragments from the DNA encoding the monoclonal antibody and subcloning the appropriate V regions into an appropriate expression vector according to standard methods. can. Phage display and aptamer technologies have been described in the literature and allow in vitro clonal amplification of very high affinity, low cross-reactivity, target-specific binding reagents. Phage display reagents and systems are commercially available from Amersham Pharmacia Biotech, Inc.; pSKAN Phagemid Display System available from MoBiTec, LLC (Marco Island, FL), and the like. Aptamer technology is described, for example and without limitation, in US Pat. Nos. 5,270,163, 5,475,096, 5,840,867 and 6,544,776.

Optionally, the one or more modified amino acid residues are glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids attached to lipid moieties, and amino acids attached to organic derivatizing agents; selected from the group consisting of A CCR5-interacting peptide may contain at least one N-linked sugar, and may contain two, three or more N-linked sugars. Peptides may also include O-linked sugars. CCR5-interacting peptides or agents can be produced in a variety of cell lines that glycosylate the protein in a manner suitable for patient use, including modified insect or yeast cells, and COS, CHO, HEK cells. cells, mammalian cells such as NSO cells, and the like. In some embodiments, the CCR5 peptide is glycosylated and has a glycosylation pattern obtained from a Chinese Hamster Ovary cell line. In most embodiments, the CCR5 interacting agent is synthesized and moieties added using techniques known in the art.

In some embodiments, the CCR5-interacting agents of the present invention are about 0.5, 1, 2, 3, 4, 6, 12, 24, 36, 48, in mammals (e.g., mice or humans) or has a half-life of 72 hours. Alternatively, they are about 0.5, 1, 2, 3, 4, 5, 6, 8, 10 in mammals (e.g. mice or humans) depending on the characteristics of the conjugate and carrier and the mode of administration. , 12, 14, 20, 25, or 30 days. In some embodiments, peptides are modified to maximize retention in muscle tissue and avoid or minimize systemic circulation. Agents can be administered in a range of retention enhancing compositions known in the art, such as gels, foams, adhesives, hydrogels, patches, and films.

Peptide size can be modified to alter its hydrodynamic radius and renal clearance. PEGylation and linker lipidation are well-established modifications that increase the serum half-life of drugs by decreasing clearance and protecting against proteases. Second generation PEGylation processes introduce the use of branched structures and alternative chemistries for PEG attachment. In particular, PEG with a cysteine-reactive group such as maleimide or iodoacetamide can allow PEGylation to a single residue within the peptide, reducing heterogeneity of the final product. Additionally, biodegradable hydrophilic amino acid polymers that are functional analogs of PEG have been developed, including XTEN (see US Patent Application No. 2019/0083577) and PAS, which is homogeneous and readily produced. Chemical conjugation of antibodies and peptides, such as that developed by ConX, is a broad description of a hybrid peptide half-life extension method that promises to overcome many of the shortcomings of previous methods.

Excipients for oral use and solubilization in injection solutions include water-soluble organic solvents (polyethylene glycol 300, polyethylene glycol 400, ethanol, propylene glycol, glycerin, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylsulfoxide). , nonionic surfactants (Cremophor EL, Cremophor RH 40, Cremophor RH 60, d-α-tocopherol polyethylene glycol 1000 succinate, polysorbate 20, polysorbate 80, solutol HS15, sorbitan monooleate, poloxamer 407, Labrafil M-1944CS, Labrafil M. - mono- and di-fatty acid esters of 2125CS, Labraol, Gellucire 44/14, Softigen 767 and PEG 300, 400 or 1750), water-insoluble lipids (castor, corn, cottonseed, olive, peanut, peppermint, safflower, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, medium chain triglycerides of coconut and palm seed oils), organic liquids/semisolids (beeswax, d-α-tocopherol, oleic acid, medium chain mono- and di- chain tri-tri-glycerides, medium-chain mono-diglycerides), various cyclodextrins (α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin), etc., and phospholipids ( hydrogenated soybean phosphatidylcholine, distearoylphosphatidylglycerol, L-α-dimyristoylphosphatidylcholine, L-α-dimyristoylphosphatidylglycerol). Chemical approaches to solubilize drugs for oral and injectable use include pH adjustment, cosolvents, complexation, microemulsions, self-emulsifying drug delivery systems, micelles, liposomes, emulsions, and the like.

Constructs/Vectors Constructs or vectors for expressing a CCR5 binding agent from recipient cells can include one or more DNA regions comprising a promoter operably linked to the nucleotide sequence encoding the peptide. Promoters can be inducible or constitutive. Examples of suitable constitutive promoters include, for example, immediate early cytomegalovirus (CMV) promoter, elongation growth factor-la (EF-la) gene promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus ( MMTV) promoter, human immunodeficiency virus (HIV) long repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and actin promoter, myosin promoter, hemoglobin promoter and creatine Human gene promoters such as kinase promoters include, but are not limited to. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, tetracycline promoters, and the like.

Expression constructs are made by any suitable method, including recombinant or synthetic techniques, utilizing a wide variety of vectors known and available in the art, including plasmids, bacteriophages, baculoviruses, mammalian viruses, and artificial chromosomes. be able to. Expression constructs may be circular or linear and should be suitable for replication and integration into eukaryotes. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses and lentiviruses. A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene transfer systems. A gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's stem cells. Many retroviral systems are known in the art.

In certain embodiments of the invention, when the peptide is provided as nucleic acid encoding the peptide, the nucleic acid is constructed as part of a suitable nucleic acid expression vector and administered so that it is present in a cell ( for example, by using retroviral vectors, direct injection, using microparticle bombardment, coating with lipids or cell surface receptors or transfection agents, or administration by linkage with homeobox-like peptides or other intracellular targeting sites. , stimulates expression of the encoded protein when administered in vivo. Alternatively, the nucleic acid can be introduced intracellularly and integrated into the host cell's DNA for expression.

The terms "nucleic acid" and "polynucleotide", used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. be They include single-, double-, and multiple-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and purine bases, pyrimidine bases, or other natural, chemical, biochemical, or chemical modifications. Included are polymers containing modified, non-natural, or derivatized nucleotide bases.

A nucleic acid is said to have a "5' end" and a "3' end" because in reacting mononucleotides to make oligonucleotides, the 5' phosphate of the pentose ring of a mononucleotide 'Oxygen is bound in one direction via a phosphodiester bond. The end of an oligonucleotide is called the "5' end" if its 5' phosphate is not attached to the 3' oxygen of the mononucleotide pentose ring. The terminus of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to the 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence can be said to have a 5' end and a 3' end even when it is contained within a larger oligonucleotide. In a linear or circular DNA molecule, an individual element is said to be "downstream" or "upstream" or 5' to the 3' element.

"Codon optimization" may also be used, in which at least one codon of the native sequence is used more or most frequently in the host cell's genes while maintaining the native amino acid sequence. Included is the process of modifying a nucleic acid sequence to enhance expression in a particular host cell by substituting codons. For example, Cas protein-encoding nucleic acids can be isolated from a given prokaryotic or eukaryotic cell, including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, mouse cells, rat cells, hamster cells, or any other host cell. Modifications can be made to replace codons with higher frequency of use in nuclear cells compared to naturally occurring nucleic acid sequences. Codon usage tables are readily available, for example, in the "Codon Usage Database". These tables can be adapted in many ways. See Nakamura et al. (2000) Nucleic Acids Research 28: 292, which is incorporated herein by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available (see, eg, Gene Forge).

The nucleic acid molecules described herein can be in any form, such as DNA or RNA, including in vitro transcribed RNA or synthetic RNA. Nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced molecules, chemically synthesized molecules, and modified forms thereof. Nucleic acid molecules can be single- or double-stranded, linear, or covalently closed to form a circle. RNA may be modified by stabilizing sequences, capping, and polyadenylation. RNA or DNA may be provided as a plasmid to express the peptide. RNA-based approaches are routinely available.

The term "RNA" refers to a molecule consisting of ribonucleotide residues, preferably consisting entirely or substantially of ribonucleotide residues. A "ribonucleotide" is a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as additions, deletions, substitutions of one or more nucleotides. and/or modified RNA that differs from naturally-occurring RNA by alteration. Such alterations can include the addition of non-nucleotide material at the ends of the RNA or internally, eg, at one or more nucleotides of the RNA. The nucleotides of RNA molecules can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs or analogues of naturally occurring RNAs.

Optimized mRNA-based compositions include 5' and 3' untranslated regions (5'-UTR, 3'-UTR) that optimize translation efficiency and intracellular stability, as known in the art. can include In one embodiment, removal of uncapped 5'-3 phosphates can be performed by treating the RNA with a phosphatase. RNA may have modified ribonucleotides to increase its stability and/or reduce cytotoxicity. For example, in one embodiment, 5-methylcytidine partially or fully replaces cytidine in RNA. In one embodiment, the term "modification" refers to providing an RNA with a 5'-cap or 5'-cap analog. The term "5'-cap" refers to the cap structure found at the 5'-end of mRNA molecules and generally consists of guanosine nucleotides linked to the mRNA via an unusual 5' to 5' triphosphate linkage. . In one embodiment, the guanosine is methylated at position 7. The term “conventional 5′-cap” refers to the 5′-cap of naturally occurring RNA, preferably the 7-methylguanosine cap. The term "5'-cap" includes 5'-cap analogs that have been modified to resemble RNA cap structures and have the ability to stabilize RNA and/or enhance translation of RNA. Providing a 5'-cap or 5'-cap analog to RNA may be performed by in vitro transcription of a DNA template in the presence of said 5'-cap or 5'-cap analog, wherein said 5'-cap is co-transcriptionally incorporated into the resulting RNA strand, or the RNA is generated, for example, by in vitro transcription and the 5'-cap is added to the RNA post-transcriptionally using a capping enzyme, such as the vaccinia virus capping enzyme. good too.

Further modifications of the RNA include elongation or truncation of the naturally occurring poly(A) tail, or modification of the 5'- or 3'-untranslated region (UTR), such as introduction of UTRs unrelated to the coding region of said RNA; For example, this may be done by replacing or inserting an existing 3'-UTR with one or more, preferably two copies of a 3'-UTR from a globin gene such as α2 globin, α globin, β globin. RNAs with unmasked poly A sequences are translated more efficiently than RNAs with masked poly A sequences. preferably with a poly-A sequence having a length of 10-500, more preferably 30-300, more preferably 65-200, especially 100-150 adenosine residues in order to increase the stability and/or expression of RNA can be modified to exist in To increase RNA expression, modifications within the coding region can be made to increase GC-content to enhance mRNA stability, codon optimization, and thus facilitate translation within the cell. The modified mRNA can be enzymatically synthesized, packaged into nanoparticles, such as lipid nanoparticles, and administered intramuscularly, for example.

Nucleic acid molecules are encapsulated in microcapsules, colloidal drug delivery systems (e.g., liposomes, microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions prepared, for example, by coacervation techniques or by interfacial polymerization. can do. Such techniques are known in the art and disclosed in Remington, the Science and Practice of Pharmacy, 20th Edition, Remington, J., ed. (2000). Target-specific delivery of drugs to particular cell subsets can improve the therapeutic index. Antibody-targeted agents that bind to cells that contain the antigen recognized by the antibody or binding fragment thereof. This includes, for example, combining maleimide-functionalized PEG-PLGA polymeric nanoparticles, or simply CCR5-interacting peptides, with a composition comprising a delivery moiety or shuttle agent.

Ex vivo approaches contemplate the application of gene editing, such as CRISPR components, to modify cells to contain or express CCR5 interacting agents as described herein.

Small molecule agonist SMAs are developed using techniques described herein or known in the art. Small molecules are further less than 1000 nM, less than 500 nM, less than 250 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 25 nM, less than 20 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM can be selected to activate the CCR5 receptor with an ED50 of In one embodiment, the CCR5-interacting agonist is selective for CCR5 or satellite cell CCR5.

Administration In accordance with the present disclosure, compositions or agents comprising or encoding a CCR5 binding agent disclosed herein may be used to improve wound healing in a variety of conditions associated with muscle loss or reduced ability to regenerate functionally. or to slow, maintain, or regenerate muscle.

The composition can be delivered by injection, by topical or mucosal application, by inhalation, or by oral routes, including modified release modes, in amounts effective to stimulate levels of muscle regeneration in a subject over a predetermined period of time. Administration can be carried out locally or systemically (eg parenterally via intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular routes) or target-specifically. In one embodiment, administration of the CCR5-interacting agent is systemic or direct to the wound. Subcutaneous or intramuscular routes may be direct administration to the affected muscle tissue.

A CCR5 interacting agent can be formulated in the form of an ointment, cream, patch, powder, or other formulation suitable for topical formulation. Low molecular weight CCR5 agonist formulations can deliver the drug through the skin and into deeper muscle tissue. Such formulations may therefore contain one or more agents that enhance the penetration of the active ingredient through the skin. For topical application, the CCR5 agent may be included in a wound dressing and/or skin covering composition.

The amount of drug to be administered can be determined by routine clinical techniques by those skilled in the art. In addition, in vitro assays may be employed to identify optimal dosage ranges. The precise dosage to be employed will depend on the nature of the drug and other clinical factors [condition of subject, body weight, age, other conditions, route of administration and type of composition (cellular, scaffold, hydrogel or oral formulation). ), etc.]. Precise dosages that are therapeutically or prophylactically effective and not harmful can be determined by those skilled in the art. Pharmaceutical compositions are suitably prepared according to conventional pharmaceutical compounding techniques. See, eg, Remington, the Science and Practice of Pharmacy, 20th Edition, Remington, J. ed. (2000) and subsequent revisions.

Reference to an effective amount includes a therapeutically or physiologically or regeneratively effective amount. As used herein, a "therapeutically effective amount" is an amount effective, at a reasonable benefit/risk ratio applicable to any medical treatment, to produce some desired therapeutic effect in at least a subpopulation of cells of an animal. , means the amount of the composition that contains chemokine receptor agonist activity. For example, the amount of CCR5 agonist administered to a subject is an amount sufficient to result in statistically significant measurable muscle repair or regeneration. Determination of a therapeutically effective amount is within the skill of those in the art. In general, a therapeutically effective amount may vary with the subject's medical history, age, condition, sex, and the severity and type of the subject's condition, as well as administration of other pharmaceutically active agents.

As used herein, the term "administration" refers to placing a composition into a subject by a method or route that results in at least partial localization of the composition at a desired site so as to produce a desired effect. means to Suitable routes of administration for the compositions of the invention depend on their format and include both local and systemic administration. In general, local administration results in more cells at a particular site being treated by delivery of the CCR5 agonist or CCR5 agonist activity compared to systemic administration to the subject, whereas systemic administration results in essentially body mass of the subject. Provides delivery to the whole. One method of local administration is by intramuscular injection.

The term "administering" according to the invention also includes transplantation of cells into a subject. As used herein, the term "transplantation" refers to the process of transplanting or transferring at least one cell to a subject. The term "transplantation" includes, e.g., autotransplantation [removal and transplantation of cell(s) from one location in a patient to the same or another location in the same patient], allograft (transplantation between members of the same species) , and xenotransplantation (transplantation between members of different species). Those skilled in the art are well aware of methods for stem cell transplantation or engraftment for muscle repair and regeneration that can be practiced in the present invention. See, eg, US Pat. No. 7,592,174 and US Patent Application Publication No. 2005/0249731, the contents of both of which are incorporated herein by reference.

As described herein, regeneration of muscle tissue by this method is associated with minimal fibrosis. Specifically, the methods and agents described herein reduce and/or inhibit the formation of scar-like tissue in damaged or non-regenerating or atrophied muscle tissue. Thus, in some embodiments, the formation of scar-like tissue in injured muscle tissue is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, compared to a control without the agent. %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. Fat deposition may be reduced as well.

However, suitable dosage ranges for intravenous administration of the peptides of the invention are generally about 1.25-5 μg of active compound per kilogram (kg) of body weight. Suitable dosage ranges for nasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5-10% by weight; oral compositions preferably contain 10-95% active ingredient.

A "derivative" is a modification of the amino acid sequence derived from the base sequence of NAMPT or containing a cif motif, or conjugation with, for example, other chemical moieties, complexation, expression (e.g., as a fusion protein), or It is meant a drug or active agent due to post-translational modification techniques as would be understood by those skilled in the art. Also included within the scope of the term "derivative" are modifications made to the parental sequence, including additions or deletions that provide functionally equivalent or functionally enhanced molecules.

"Isolated" means material that is substantially or essentially free from components that normally accompany it in its original state.

The term "subject" refers to any medical or veterinary subject, including patients. A subject can be a vertebrate subject, such as a mammalian subject (eg, bovine, porcine, canine, feline, equine, llama, camelid, etc.), non-mammal, reptilian avian, fish. Subjects include humans for whom prevention or treatment is desired. A subject may be in need of prophylaxis or treatment for cancer, wound healing, sarcopenia, or other pathology, disease, disorder or condition associated with tissue degeneration.

The term "polynucleotide" or "nucleic acid" as used herein refers to mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to oligonucleotides greater than 30 nucleotides in length.

As used herein, the term "identity" of sequences refers to the extent to which sequences are identical, either nucleotide-wise or amino acid-wise, over the window of comparison. Thus, "percentage of sequence identity" compares two sequences that are optimally aligned over the window of comparison, and includes identical nucleobases (e.g., A, T, C, G, U) or identical amino acid residues. Groups such as Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met appear in both sequences. It can be obtained by counting the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiplying the result by 100. For the purposes of the present invention, "sequence identity" is used by the DNASIS computer program (Version 2.5 for Windows, available from Hitachi Soft Engineering Co., Ltd., South San Francisco, CA, USA) and in the reference manual accompanying the software. It can be understood to mean "percentage match" calculated using standard defaults as specified. Amino acid sequence identity may also be determined using the EMBOSS Pairwise Alignment Algorithms tool available from The European Bioinformatics Institute (EMBL-EBI), part of the European Molecular Biology Laboratory. This tool is available at www. ebi. ac. It can be accessed from the website at uk/Tools/emboss/align/. This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). The default settings are Gap Open: 10.0 and Gap Extend 0.5. The default matrix "Blosum62" is used for amino acid sequences.

The term "similarity" of sequences refers to the percentage number of amino acids that are identical or constitute conservative amino acid substitutions, as defined in Table 1 below. Similarity can be determined using a sequence comparison program such as GAP (Deveraux et al, 1984 Nucleic Acids Research 12:387-395). In this way, sequences of similar or substantially different lengths to those cited herein are made comparable by inserting gaps into the alignment, and such gaps are used, for example, by GAP. determined by a comparison algorithm. Methods are described herein that involve conventional molecular biology techniques. Such techniques are generally known in the art, see Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2001), and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, (1992) (including periodic revisions). ing. Immunology techniques are generally known in the art, see Current Protocols in Immunology, ed. Coligan et al., Greene Publishing and Wiley-Interscience, New York, (1992) (with periodic revisions); Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, Mass., (2007); Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, Boca Raton, Fl, (2006); Medical Immunology, 6th ed., edited by Gabriel Virella, Informa Healthcare Press, London, England, (2007), and Harlow and Lane ANTIBODIES: A Laboratory Manual, Second edition Cold Spring Harbor Laboratory Press , Cold Spring Harbor, N.Y., (2014). Conventional methods of gene transfer and gene therapy can also be adapted for use in the present invention. See, for example, Gene Therapy: Principles and Applications, ed. T. Blankenstein, Springer Verlag, 1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed. P. D. Robbins, Humana Press, 1997; Viral Vectors for Gene Therapy: Methods and Protocols, ed. Otto-Wilhelm Merten and Mohammed Al-Rubeai, Humana Press, 2011, and Nonviral Vectors for Gene Therapy: Methods and Protocols, ed. Mark A. Findeis, Humana Press, 2010. Amino Acids. 2018 Jan; 50(1) doi: 10.1007/s00726-017-2516-0. See Epub 2017 Nov 28.

References detailing the methods employed herein are listed below.
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This application discloses, but is not limited to, the following methods useful in practicing the described invention.

Zebrafish Strains and Maintenance The extant transgenic lines used were TgBAC(pax3a:GFP) ⁱ¹⁵⁰ [referred to as TgBAC(pax3a:GFP)] ⁴⁰ , Tg(mpeg1:mCherry) ^gl23 [Tg(mpeg1:mCherry) and ⁴¹ , Tg (mpeg1: GAL4FF) ^gl25 [referred to as Tg (mpeg1: GAL4FF)] ⁴¹ , Tg (UAS-E1b: Kaede) ^s1999t [referred to as Tg (UAS: Kaede)] ⁴² , Tg (UAS-E1b: Eco.NfsB-mCherry) ^c264 [referred to as Tg (UAS: NfsB-mcherry)] ⁴³ , Tg (−8mpx: KALTA4) ^gl28 [Tg (mpx: KALTA4)] ^{44, 45} , Tg (referred to as actc1b: EBFP2) ^pc5 [referred to as Tg (actc1:BFP)] ⁴⁶ , Tg (ubi: secAnnexinV-mVenus) ^mq8Tg [referred to as Tg (ubi: secAnnexinV-mVenus)] ⁴⁷ , Tg (actc1b: GFP) ^zf10 [referred to as Tg (actc1b: GFP) ⁴⁸ . All experiments were performed according to Monash University guidelines and approved by the local ethics committee. All procedures using animals at the Hubrecht Institute were approved by the local Animal Care and Use Committee and were performed in accordance with national and European legislation and in compliance with animal welfare laws, guidelines and policies. Staging and rearing were performed as previously reported [Westerfield, M. The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio). (University of Oregon press, 2007)] ⁵¹ . All embryos were maintained at 28.5° C. in Ringer's solution and treated with 8 hpf to 0.003% 1-phenyl-2-thiourea (PTU) (Sigma-Aldrich).

Generation and Genotyping of Zebrafish Mutant Lines Mutations of nampta and ccr5 were generated using the CRISPR/Cas9 system. Synthetic guide RNA (gRNA) targeting the gene of interest was generated as a crRNA:tracrRNA duplex [Alt-R® CRISPR-Cas9 system, IDT]. Gene-specific crRNA sequences were selected using the Alt-R® CRISPR-Cas9 Custom Guide RNA Design Tool (IDT) (nampta crRNA: 5′-acgacacaagacggtcttatGGG-3′, ccr5 crRNA_1: 5′- gtagcacccatgcaacaaTGG-3', ccr5 crRNA_2:5'-attttcctgataatacatccTGG-3'). Gene-specific crRNA was heteroduplexed to universal tracrRNA according to the manufacturer's recommendations to generate double-stranded gRNA. gRNA [nampta mutant with single gRNA, ccr5 mutant with double gRNA (ccr5 crRNA_1+cr5 crRNA_2)] and recombinant Cas9 protein [Alt-R® S. pCas9 Nuclease, IDT] was injected into the scutellum of 1-cell stage wild-type embryos to generate mutants. Injected embryos were grown to adulthood, mated with wild-type zebrafish, and screened to identify founders containing germline mutations. The mutant of interest identified is nampta c. 180_182delinsTCCGTCTTGCTGACCTTTCCAGCAG (p.Try61Profs*4) (referred to as namptapc41) and ccr5 c. 66578 delins ACCCCTATGCAACATCATTTACCAATGAGCAAATGGATTTAAACAAGAAATCCTGCCAACTTGATTTTCCTGATAATACATAATA (p.Pro24Leufs*28) (referred to as ccr5 ^pc42 ). For genotyping of nampta mutants, DNA was isolated from excised fins (adults) or whole embryos and oligonucleotides designated Nampta_F (5′-tgccgtgagaagacaga-3′) and Nampta_R (5′-gcaatcaattgccttacctttt-3′) were generated. (PCR product size, nampla 117 bp and nampta ^pc41 141 bp). For genotyping of ccr5 mutants, PCR was performed with oligonucleotides Ccr5_F (5′-aacgaaaactgggcatgtagc-3′) and Ccr5_R (5′-ccgggaataacaaagctca-3′) (PCR product size, ccr5 618 bp and ccr5 ^pc42 173 bp).

Muscle injury in zebrafish larvae 4 dpf larvae were anesthetized with 0.01% tricaine (MS-222) (Sigma-Aldrich) in Ringer's solution. Mechanical injury was aimed at either the dorsal or ventral muscle layer above the vulva when larvae were oriented dorsally to the top and anterior to the left. Needlestick injury was performed as previously reported [Gurevich, DB et al. Asymmetric division of clonal muscle stem cells co-ordinate muscle regeneration in vivo. Science 353 6295 aad9969 (2016)]. Briefly, a single 30-gauge needle puncture into the muscle was performed, producing extensive damage to many damaged muscle fibers. For laser-induced injury, anesthetized larvae were mounted in a thin layer of 1% low melting point agarose in Ringer's solution. Injury was performed using a UV-nitrogen laser pulsed through a coumarin 440 nm dye cell connected to a Zeiss Axioplan microscope (MicroPoint Laser System, Andor Technology). Laser damage required an average 5-10 second pulse from the laser beam focused through a 40X water immersion objective. For time-series analysis of the immediate response to injury, an Olympus FVMPE-RS upright multiphoton microscope equipped with a 25X/1.05 water immersion objective was used with a SIM scanner (Olympus) at 790 nm, 200 msec dwell time. , muscle fiber ablation was performed at 100% laser power. Macrophages responding to injury were tracked using Fiji's manual tracking plugin.

Evaluation of Volumetric Muscle Loss Injury and Repair in Mice Injury: Male C57BL/6J mice aged 10-12 weeks were anesthetized and the left hind limb was shaved. A unilateral incision of approximately 1 cm was made to expose the underlying fascia. The left hind limb was extended and retracted as it exited through the incision site. A 3×4 mm full thickness resection of the rectus femoris muscle was excised. Immediately thereafter, 200 ng or 500 ng of hrNAMPT ₍₁₎ [hydrogel component; /ml aprotinin (ab146286, Abcam)] was filled and polymerized at the defect. The soft tissue was then sutured closed.

Histological Analysis: Ten days after treatment, animals were sacrificed and wounds were harvested for histological analysis. The defect site and associated proximal and distal segments of the quadriceps (including the rectus femoris, vastus medialis, and vastus lateralis) were resected and embedded. Histological analysis was performed on serial paraffin sections (4 μm sections taken through the central portion of the wound). Multiple sections were stained with Masson's trichrome (to detect collagen deposition) and the degree of fibrosis (represented by blue staining) was evaluated using ImageJ software (version 1.51h, National Institutes of Health, USA). Measured by histomorphometric analysis. To maintain sample-to-sample uniformity, vastus medialis lengths taken at multiple depths ranging from 1.0 mm to 3.0 mm were used as a reference between tissue sections to determine depth during section preparation. board. For fibrosis quantification, the average muscle fibrosis area at each depth was scored and normalized by the area of the rectus femoris. The total muscle area is determined by calculating the average area of the rectus femoris muscle at each depth.

Immune cell profiling and PAX7 ⁺ cell quantification by flow cytometry. Mice were euthanized by _CO2 asphyxiation 4, 6 or 8 days after delivery of 0.5 μg hrNAMPT ₍₁₎ by fibrin hydrogel or treatment with control fibrin hydrogel alone. The defect site and associated quadriceps proximal and distal segments were isolated and placed in 890 μl of complete RPMI (with 10% FBS and 2 mM Glutamax, Life Technologies). The tissue was minced with surgical scissors and added with 100 μl of 10 mg/ml collagenase II (Sigma-Aldrich) and 10 μl of 10 mg/ml DNAse I (Biolabs) and 100 μl of dispase II (10 mg/ml) for PAX7 acquisition. was added to the digest for The mixture was vortexed and incubated at 37°C for 45 minutes. Collagenase was then inactivated with 500 μl ice-cold PBS, 5% FBS, 5 mM EDTA. The mixture was filtered through 70 μm and 40 μm filters. The supernatant was discarded and the pellet was resuspended in 250 μl of complete RPMI, dispensed into wells of a 96-well U-bottom plate and used for antibody staining. The cell solution was centrifuged, the supernatant discarded and washed with PBS. The cell viability stain used was 100 μl of Zombie Aqua (Biolegend) Live-Dead dye diluted in PBS (1:400 dilution) and incubated at 4° C. for 30 minutes. Cells were then blocked with FcX (anti-CD16/32 antibody, Biolegend, 1 μg/ml) flow cytometry buffer (PBS, 5% FBS). After holding at 4°C for 20 minutes, it was washed with flow cytometry buffer and centrifuged. Primary surface antibody staining was performed in duplicate with 100 μl anti-mouse antibody cocktail (Biolegend) diluted in flow cytometry buffer. T cell staining was performed with anti-CD4 (clone RM4.5, #100516), anti-CD8 (clone 53-6.7, #100738), and anti-CD3 (clone 17A2, #100220) at 2 μg/ml. Neutrophils and macrophages were treated with 2 μg/ml anti-CD11b (clone M1/70, #101208), 1 μg/ml anti-Ly6G (clone 1A8, #127628), 4 μg/ml anti-F4/80 (clone BM8, #101208). 123147), 10 μg/ml anti-CD80 (clone 16-10A1, #104714), and 2.6 μg/ml anti-CD206 (clone C068C2, #141720). Cells were stained on ice for 30 minutes and washed as above. For internal Foxp3 staining of the T cell panel, cells were fixed with 100 μl fixation/permeabilization solution (42080, Biolegend) for 35 minutes. Cells were then washed and resuspended in 100 μl flow cytometry buffer containing 0.5% saponin and 5 μg/ml anti-Foxp3 (clone 3G3, #35-5773-U100) for 45 minutes. Cells were then resuspended in flow cytometry buffer (100 μl) and harvested on a Fortessa x20 (Beckman Coulter). Flow cytometry staining of satellite cells was performed with 200 μl of antibody cocktail (Biolegend) diluted in flow cytometry buffer. 5 μg/ml anti-VCAM/CD106 biotin (clone 429 (MVCAM.A), #105703), 2.5 μg/ml anti-streptavidin (#405250), 2 μg/ml anti-CD45 (clone 30-F11, #103114 ), anti-CD11b (clone M1/70, #101208), anti-Ly6G (clone 1A8, #127607), 1 μg/ml anti-CD31 (clone MEC13.3, #102507) were added. Cells were stained on ice for 45 minutes and washed as above. Cells were also stained for 1 hour on ice with 200 μl flow cytometry buffer with 0.5% saponin containing the following intracellular antibody cocktail: Biolegend 1 μg/ml anti-Ki67 (clone 16A8, #652411), Novus Biologicals 10 μg/ml. ml Anti-Pax7 (clone Pax7/497, #NBP2-34706AF488). Cells were then resuspended in flow cytometry buffer (275 μl) supplemented with 25 μl of Invitrogen Count Bright Absolute Counting Beads (25,000 beads, #C36950) and acquired on a Fortessa x20 (Beckman Coulter). All events were acquired, and the number of PAX7 ⁺ cells per 10,000 damaged cells was calculated by the following formula: PAX7 ⁺ number in the injured area = 10,000 × number of PAX7 ⁺ cells / [(1/25,000) × number of beads×(percentage of viable cells/100)×total number of cells after digestion]. The same calculation was performed to quantify the number of proliferating PAX7 ⁺ cells using the number of PAX7 and Ki67 double positive cells.

Immunofluorescence staining of frozen sections: Immunostaining was performed on 10 μm cryosections using a standard protocol (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) with antigen retrieval. Cutting pieces are 2 % BSA, 5 % NORMAL GOAT SERUM / PBS, 0.3 % Triton -X and AffiniPure Fab Fab Fab Fab Fab Fab Fab Fab Fab FAB FABAT GOAT ANTI -MOUSE IGG (H + L). Blocked with MUNO RESEARCH LABORATORIES) and mouse antibodies for mouse tissue Non-specific binding was minimized. Antibodies: Mouse anti-mouse Pax7 (2 μg/ml, Development Studies Hybridoma Bank) and secondary Alexa Fluor-coupled antibody (Thermo Fisher) were used. Muscular sarcholema was visualized with rhodamine-labeled wheat germ agglutinin (WGA) (Vector Laboratories) and nuclei with DAPI (Sigma-Aldrich) staining.

Quantification of muscle fibers with central nuclei: Hematoxylin and eosin (H&E) staining was performed on 4 μm paraffin-embedded sections. The number of central nuclei within muscle fibers was counted from 5 serial sections per sample by histomorphological analysis using ImageJ software (version 1.51h, National Institutes of Health). To maintain uniformity between samples, the length of the vastus medialis muscle taken at multiple depths ranging from 1 mm to 3 mm was used as a reference between tissue sections to determine depth during section preparation. To quantify the average number of central nuclear cells, the total number of nuclei at each depth was normalized by the area of the rectus femoris.

Microscopy and Image Analysis Time-series imaging of whole larvae to track macrophages in response to injury was described in Zeiss Lightsheet Z.; 1 microscope and 5X/0.16 air objective, ambient control (28.5°C). The XY resolution was 1.14 μm, the Z resolution was 5.5 μm, and the thickness of the light sheet was 11.68 μm. Total imaging time per larva was 25 h (1000 3D stacks acquired at 1.5 min intervals) and larval viability was determined by assessing heart rate at the end of the imaging session. For tracking, macrophage images were first filtered with a 3D median filter and segmented with a hysteresis threshold using the 3D ImageJ Suite ⁵³ algorithm. Low and high hysteresis thresholds were selected visually. The dataset was then time-reversed to track cells exiting the injury site. Wound edges were labeled manually in ImageJ. The tracking procedure was based on overlapping segmentation of cells between consecutive frames. Cases where two cells were too close together to form one object were marked as 'merged' because the segmentation algorithm was unable to separate them. Tracked macrophage images were inverted for visualization of results.

Line scanning confocal microscopy for long-term time-series imaging and single Z-stack acquisition was performed using a Zeiss LSM 710 upright confocal microscope equipped with a 20X/1.0 water immersion objective. Photoelectric conversion was performed using a bleaching tool with a 405 nm diode laser.

Time-series imaging with high temporal and spatial resolution was performed using an inverted LSM 880 fast AiryScan confocal equipped with a 40X/1.3 oil immersion objective and piezo Z-stage. The voxel size was constant at 0.2×0.2×1 μm, and the frame rate was 3-18 frames/sec depending on the field of view. Photobleaching was measured after imaging and was determined to be minimal for imaging times up to 1 hour.

Fixed and immunostained cell culture samples were imaged on a Leica DMi8 inverted widefield microscope using a 10× objective. Birefringence imaging was performed as previously reported [Berger, J., Sztal, T. & Currie, P.D. Quantification of birefringence readily measures the level of muscle damage in zebrafish. 785-788 (2012)], using a Leica DM IRB upright microscope integrated with Abrio software (CRI Hinds Instruments) and using a 5X objective. Images were analyzed using the software Fiji52. This normalized the mean gray value of the birefringence of the injury site to the regions before and after it to calculate the relative birefringence of the wound site.

Microscopic images were processed with Adobe Creative Cloud 2018, Fiji ⁵² and Imaris 9.2 (Bitplane). Counting of macrophage numbers and further 3D analysis were performed by surface rendering the volume using Imaris. The sphericity analysis evaluated the deviation of the cell shape from a perfect sphere (assigned an arbitrary value of 1). Circularity values were generated as summary statistics of surface rendering. Proliferating stem cell counts were performed in Fiji. Acquisition channels for PAX7 and EdU were segmented using threshold commands. The image calculator generated a mask channel of EDU-positive PAX7 cells only. Cell numbers were counted using the Analyze Particles command.

Chemical Treatment Cell ablation was performed as previously reported [Pisharath, H. & Parsons, MJ in Zebrafish 133-143 (Springer, 2009)] with minor modifications. Appropriate stage larvae were incubated in 5-10 mM metronidazole (Mtz) (Sigma-Aldrich) in Ringer's solution and refreshed daily until experimental endpoint. Drug treatment was achieved by incubating 4 dpf needlestick-injured larvae with solutions in Ringer's solution of 5 and 10 μM Senikriviroc (CVC) (Med Chem Express) and 5 and 10 μM Maraviroc (MVC) (Med Chem Express) immediately after injury and refreshed daily. It was implemented by

The effect of drug treatment on muscle damage by laser ablation was assessed by treating larvae with 5 μM CVC for 2 hours prior to injury and maintaining drug until experimental endpoint. For NAMPT enzyme inhibition experiments, 5 dpf/1.75 dpi needlestick-injured larvae were transferred to Ringer's solution containing 10 μM GMX1778 (Sigma-Aldrich) and maintained there until the experimental endpoint. Chemokine supplementation experiments were performed overnight on plasticware coated with 0.1% bovine serum albumin (BSA) (rinsed with Ringer's solution before use) to minimize protein adsorption. Larvae were treated with 57 nM recombinant human visfatin [hrNAMPT(1)] (PeproTech), 57 nM recombinant mouse CCL8/MCP-2 (mrCCL8) (PeproTech), Mtz in combination with CVC.

Supplementation of drugs to cell cultures was performed by adding the following to the growth medium of C2C12 cells for 6 hours: 1.9, 9.5 and 19 nM hrNAMPT ₍₁₎ (PeproTech), 1.9 and 9.nM. 5 nM recombinant human visfatin [hrNAMPT ₍₂₎ ] (Enzo Life Sciences), 100 nM CVC (Med Chem Express), 100 nM MVC (Med Chem Express), 100 nM PF-4136309 (PF4) (Med Chem Express), 9. 5 nM mrCCL8, 9.5 nM recombinant mouse CCL4/MIP-1β (mrCCL4) (PeproTech), 9.5 nM recombinant mouse CCL2/MCP-1 ((PeproTech), 500 nM GMX1778 (Sigma-Aldrich) and hrNAMPT ₍₁ The following were added to the growth medium of mouse primary myoblast co _- cultures for 24 hours: 9.5 nM hrNAMPT ₍₂₎ (Enzo Life Sciences), 100 nM CVC (Med Chem Express), 100 nM MVC (Med Chem Express), 100 nM PF4 (Med Chem Express) and 9.5 nM mrCCL4 (PeproTech).

LysoTracker Assay:
Larvae were incubated with 10 μM LysoTracker™ Deep Red (Thermo Fisher)/Ringer's solution for 1 hour in the dark and rinsed 5 times with fresh Ringer's solution before imaging.

5′-Ethyl-2′-deoxyuridine (EdU) labeling Larvae: Labeling and detection were performed as previously reported [Nguyen, PD et al. Muscle stem cells undergo extensive clonal drift during tissue growth via Meox1-mediated induction of G2 cell-cycle arrest. Cell Stem Cell 21, 107-119. e106 (2017)] ⁵⁵ , with minor modifications. 6 dpf/2 dpi larvae were pulsed with 2.5 nM EdU (Thermo Fisher) for 1 hour and an additional pulse of 1.5 hours before fixation. Cell culture: Cells were incubated for 1 hour in 10 μM EdU supplemented medium. After Edu pulse, C2C12 cells were immediately fixed and primary mouse myoblast co-culture cells were washed with PBS, replaced with medium and incubated for another 2 hours before fixing the cells. Cells were treated using the Click-iTTMEdU Alexa Fluro™ 647 Imaging Kit (Thermo Fisher) according to the manufacturer's protocol.

Immunohistochemistry and in situ hybridization. Antibody staining for myogenic cells was performed as previously reported [Figeac, N., Serralbo, O., Marcelle, C. & Zammit.PloS one 6, e19713 (2011)]. [Figueac, N., Serralbo, O., Marcelle, C. & Zammit, PS ErbB3 binding protein-1 (Ebp1) controls proliferation and myogenic differentiation of muscle stem cells. Developmental biology 386, 135-151 (2014)] . Following in situ hybridization, antibody staining was performed using standard procedures. Antibodies: mouse anti-Pax7 (1:10, DSHB), chicken anti-GFP antibody (1:500, Thermo Fisher), mouse anti-mCherry antibody (1:500, Abcam), rat anti-mCherry antibody (1:500, Kerafast) ( immunohistochemistry), rabbit anti-PBEF1 (anti-NAMPT) antibody (1:50, Sigma-Aldrich) and secondary Alexa Fluro-coupled antibody (Thermo Fisher). Nuclei were visualized by staining with DAPI (Sigma-Aldrich). In situ hybridization and probe preparation were performed as previously reported [Thisse, C. & Thisse, B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nature protocols 3, 59 (2008)]. Antisense probe used: mmp9 [de Vrieze, E., Sharif, F., Metz, JR, Flik, G. & Richardson, MK Matrix metalloproteinases in osteoclasts of ontogenetic and regenerating zebrafish scales. Bone 48, 704-712 (2011 )]. The nampt (ENSDARG00000030598) PCR probe containing the T7 RNA polymerase promoter 3′ to the antisense probe and the SP6 RNA polymerase promoter 5′ to the sense probe was used with primers 5′-GAGtatttaggtgacactatagGTTTCATCGCAAGACGG-3′ and 5′-GAGtaatacgactcactatagGC GGGAAGCACCTTATAGCCT-3 ' was made using Hematoxylin and eosin staining was performed on 10 μm cryostat sections of 5 dpf and 7 dpf larvae according to standard methods.

Binding of NAMPT to CCR5 (ELISA)
Binding of hrNAMPT to hrCCR5: ELISA plates (moderate binding, Greiner Bio-One) were coated overnight at 4°C with 1% BSA or 20 nM GST-fused human CCR5 (hrCCR5, Abcam) in PBS. Wells were blocked with 1% BSA in PBS containing 0.05% Tween-20 (PBS-T) for 1 hour at room temperature. Wells were washed three times with PBS-T and further incubated with hrNAMPT ₍₁₎ (Peprotech) at increasing concentrations (0 nM to 800 nM) in PBS-T containing 0.1% BSA for 1 hour. Bound NAMPT molecules were detected using a biotinylated antibody for NAMPT and HRP-streptavidin (Human PBEF/Visfatin DuoSet ELISA, R&D Systems). Using the signal obtained from the BSA-coated wells, non-specific binding at each NAMPT concentration was subtracted to calculate the amount of specific binding. Specific binding data were non-linearly regressed in Prism 7 to determine the dissociation constant (KD) using A450nm=Bmax*[NAMPT]/(KD+[NAMPT]).

Competitive binding of hrNAMPT to mrCCR5: ELISA plates (moderate binding, Greiner Bio-One) were coated overnight at 4°C with 1% BSA or 20 nM recombinant mouse CCR5 (MyBioSource) in PBS. Wells were then blocked with 1% BSA in PBS containing 0.05% Tween-20 (PBS-T) for 1 hour at room temperature. The wells were washed three times with PBS-T and then treated with increasing concentrations (0 nM to 400 nM) of mouse CCL4 (Peprotech) in PBS-T with 0.1% BSA containing 100 nM hrNAMPT ₍₁₎ (Peprotech) for 1 hour. incubated. Bound hrNAMPT ₍₁₎ molecules were detected using a biotinylated antibody for NAMPT and HRP-streptavidin (Human PBEF/Visfatin DuoSet ELISA, R&D Systems). The signal obtained from the BSA-coated wells was used to subtract the non-specific binding at each rhNAMPT concentration to calculate the amount of specific binding. The specific binding data were calculated by A ₄₅₀ nm = A ₄₅₀ nm Min + (A ₄₅₀ nm Max - A ₄₅₀ nm Min)/(1 + 10 ^{(X-Log IC50)} ) to determine the half-maximum inhibitory concentration (IC50) of CCL4 and non-linear regression with Prism 7. was fitted by

NAMPT in macrophage supernatant: Maf/DKO cells and mouse macrophage cell line Raw 264.7 (ATCC) were cultured in growth medium (DMEM + 10% FBS + 10 ng/ml M-CSF) for 16 hours, and proteins in the recovered supernatant were analyzed. , was concentrated using Amicon® Ultra-15 centrifugal filters (Merck) with a nominal molecular weight limit of 10 kDa. NAMPT in the supernatant was quantified using the Human PBEF/Visfatin DuoSet ELISA (R&D Systems) according to the manufacturer's instructions. Cell surface CCR5 receptor concentration: The mouse muscle cell line C2C12 was cultured as described above. Cells were detached using a cell scraper when 70-80% confluent and membrane proteins were isolated using an extraction kit (Plasma Membrane Protein Extraction Kit, Abcam). Next, the CCR5 concentration in the membrane extract was measured by ELISA (Mouse Ccr5 ELISA Kit, Biorbyt), and the amount of CCR5 per cell was calculated as [CCR5] _{(molecule/cell)} = [CCR5] _(ng/cell) /CCR5. Calculated using _mw * ^10-9 * _N0 (where _N0 = Avogadro's constant).

Mouse Cytokine Array Maf/DKO cells were cultured in growth medium (DMEM+10% FBS+10 ng/ml M-CSF) for 16 hours and the supernatant was harvested. Cytokine arrays (Proteome profiler array, R&D systems, ARY006) were performed according to the manufacturer's instructions. Briefly, nitrocellulose membranes were blocked for 1 hour at room temperature on a rocking platform shaker. During the blocking step, 0.7 ml samples were brought up to 1.5 ml with Array Buffer and incubated with Mouse Cytokine Array Panel A Antibody Cocktail for 1 hour at room temperature. Samples were then incubated on the membrane overnight at 4°C on a rocking platform shaker. Membranes were washed and then incubated with streptavidin-HRP for 30 minutes at room temperature. Finally, Chemi Reagent Mix was added to develop the membrane, imaged on a Biorad Chemidoc MP system and analyzed using (Image Lab software, Bio Rad).

Cell culture Mouse muscle cell line C2C12 [Yaffe, D. & Saxel, O. Serial passingaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. 5 g/l D-glucose, no L-glutamine, no pyruvate NA (Gibco)) + 20% fetal bovine serum-One Shot (Gibco) + 1% Glut Max 100x (Gibco)]. Cells were maintained at 37°C, 5% CO2. 70% confluent, passage 8 cells were extracted from T75 flasks with 0.025% trypsin EDTA (Gibco), neutralized with growth medium and spun at 180 xg for 5 minutes to pellet the cells. Cells were then resuspended in 10 ml fresh growth medium. 500 μl of cells were plated on 8-well on cover glass II (Sarstedt) chamber slides at a density of 1×10 ³ cells/ml. Cells were allowed to reattach for 4 hours at 37°C. For drug treatment, the medium was supplemented with the appropriate dose and cultured for 6 hours.

For the isolation of mouse primary myoblasts, E17.5 C57/BL6J mouse limb skeletal muscle was minced and digested with 0.125% trypsin at 37° C. for 20 minutes. Fibroblasts were depleted by plating cells for 1 hour in growth medium (DMEM + 20% FBS) in 10 ^cm2 tissue culture dishes (2 embryos per dish). Medium containing non-adherent cells was replated in growth medium on gelatin-coated 10 cm ² tissue culture dishes for 24 hours. Myoblasts were again depleted of fibroblasts prior to co-culture on gelatin-coated 48-well plates in DMEM+20% FBS+10% L929 conditioned medium. 100,000 myoblasts, 7500 MafB/c-Maf-deficient (Maf-DKO) macrophages per well [Aziz, A., Soucie, E., Sarrazin, S. & Sieweke, MH MafB/c- Maf deficiency allows self-renewal of differentiated functional macrophage. Science 326, 867-871 (2009)] or plated with 1,000 3T3 cells. For drug treatment, the medium was supplemented with the appropriate dose and cultured for 24 hours.

Cell surface CCR5 receptor concentration:
The mouse muscle cell line C2C12 was cultured as described above (see above). Mouse macrophage cell line Raw 264.7 (ATCC) was cultured in growth medium (Dulbecco's modified Eagle medium + 10% FBS). Cells were detached using a cell scraper when 70-80% confluent and membrane proteins were isolated using an extraction kit (Plasma Membrane Protein Extraction Kit, Abcam). Next, the CCR5 concentration in the membrane extract was measured by ELISA (Mouse Ccr5 ELISA Kit, Biorbyt), and the amount of CCR5 per cell was calculated as [CCR5] _{(molecule/cell)} = [CCR5] _(ng/cell) /CCR5. Calculated using _mw * ^10-9 * _N0 (where _N0 = Avogadro's constant).

Fluorescence Activated Cell Sorting (FACS), Single Cell RNA Sequencing and Analysis Wound-responsive macrophages were isolated by FACS as previously reported [Ratnayake, D. & Currie, PD in Myogenesis 245-254 ( Springer, 2019)], with the following changes: 4 dpf Tg(mpeg1:mCherry) larvae were injured with a needle stick, the injury site was dissected at 1, 2, 3 dpi and the tissue dispersed into a single cell suspension. Trunk tissue from uninjured larvae was also included in the analysis. Cells were sorted using a FACS Aria II (BD biosciences). Viable individual macrophages (based on mCherry fluorescence, DAPI exclusion, forward and side scatter properties) were spiked with 100-200 nl of CEL-seq primers, dNTPs, synthetic mRNA Spike-In in 5 μl of Vapor-Lock (Qiagen). were sorted into pre-prepared 384-well plates containing Plates were spun down immediately after sorting and frozen at −80° C. until sequencing.

Single-cell RNA-sequencing libraries were prepared using the SORT-seq platform as previously reported [Muraro, MJ et al. A single-cell transcriptome atlas of the human pancreas. Cell systems 3, 385-394. e383 (2016)]. This platform follows the Cel-Seq2 protocol [Hashimshony, T. et al. CEL-Seq2: Sensitive highly-multiplexed single-cell RNA-Seq. Genome biology 17, 77 (2016)] with the help of a robotic liquid handler. done. This protocol results in each cell being barcoded and generating a single-cell transcriptome for every macrophage isolated. Each timepoint was reproduced independently, resulting in approximately 768 macrophages (3072 total macrophages) to be sequenced individually per timepoint. Next-generation sequencing was performed using the Illumina NextSeq platform. Paired reads were mapped against the zebrafish reference assembly version 10 (GRC10). FASTQ files were processed as previously reported [Muraro, MJ et al. (2016); Gruen, D. et al. De novo prediction of stem cell identity using single-cell transcriptome data. Cell Stem Cell 19, 266-277 (2016)]. 3′ to enhance the mappability of transcripts using bwa [Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589-595 (2010)]. Using a transcriptome dataset with improved UTR annotation [Junker, JP et al. Genome-wide RNA Tomography in the zebrafish embryo. Aligned to the tome. Read 1 was used to assign reads to the correct cell or library and read 2 was mapped to the gene model. For read counting, UMI barcode correction was first performed by removing duplicate reads with the same combination of library, cell, and molecular barcode mapped to the same gene. Transcript counts were then adjusted to the expected number of molecules based on counts, 256 possible UMIs, and Poisson count statistics. The script for creating the count file is available at https://github. com/vertesy/TheCorvinas/tree/master/Python/MapAndGo2: https://github. com/vertesy/TheCorvinas/blob/master/Python/MapAndGo/Readme_MapAndGo. present in md. Spiked-in RNA was discarded and not included in further analysis. Transcript counts for all assay plates (injury-type technical replicates) were combined into one matrix for downstream analysis. Downstream analysis of transcript counts of combined samples was performed using Seurat (v2.3.4). Transcript count matrices were imported using the CreateSeuratObject function (min.cell=25, min.genes=250) and FilterCells discarded low-quality cells with the following thresholds: min 500 and max 3500 genes, max 10%. mitochondrial genes, UMI of minimum 1000 and maximum 15000). The filtered dataset was log-normalized using a scale factor of 10000 and a set of ~2800 genes was used for linear dimensionality reduction (FindVariableGenes, x.low.cutoff=0.05, x.high.cutoff=3 , y.cutoff=0.5). Unnecessary sources of variability were removed by regressing the number of UMIs per cell, the percentage of mitochondrial reads per cell. Clustering analysis is performed by Seurat's FindClusters function (reduction.type=“pca”, dims.Use=1:50, resolution=0.5, save.SNN=TRUE) and nonlinear dimensionality reduction using RunTSNE (reduction.use= "pca", dims.use=1:50, perplexity=30, tsne.method="Rtsne"). Seven cell clusters were identified. All clusters using the Find library kers(logfc.threshold=0.25, test.use=“wilcox”) or FindMarkers(min.pct=0.20) functions in the biomarker library of clusters were compared to each other or cluster-specific comparisons were made. Trajectory analysis was performed using partition-based graph abstraction (PAGA) ⁸⁶ which is part of the scanpy package (v1.4.5.2.dev6+gfa408dc) ⁸⁷ . In summary, a neighborhood graph of data points (cells) is generated using scanpy. pp. generated using the neighbors function ⁸⁸ (35 neighbors starting with the top 50 principal components as in PCA and UMAP), then scanpy's scanpy. tl. PAGA was performed on the previously identified clusters as a group using the paga function. Finally, PAGA's cell embedding is based on scanpy's scanpy. tl. It was rendered using a Force Direct Layout (FDL) initialized with PAGA coordinates using the draw_graph function (Force Atlas 2 layout).

Pseudo-time analysis was performed using diffusion pseudo-time (DPT) ⁸⁹ . Scanpy's scanpy. tl. The dpt function was run with the previously defined cluster 3 (composed of cells at uninjured timepoints) as the root. PAGA and FDL plots are from scanpy sc. pl. paga sc. pl. paga_path(n_avg=25) and scanpy. pl. Created using the draw_graph function.

Biological process enrichment analysis using differentially expressed genes in each cluster was performed using Metascape (http://metascape.org).

Zebrafish tissue-specific loss-of-function mutations Tol2-flanked transgene Tg(4xUAS:NLS-Cas9, cryaa:EGFP) ^gl36Tg [Tg(4XUAS:NLS-Cas9, cryaa:EGFP)] and Tg(4xUAS:NLS-Cas9 , cmlc2:RFP) ^gl37Tg [denoted as Tg(4XUAS:NLS-Cas9, cmlc2:RFP)] was assembled by Gateway cloning. These constructs were microinjected along with Tol2 mRNA in the required background. Tg (4XUAS: NLS-Cas9, cryaa: EGFP) is Tg (mpeg1: GAL4FF / UAS: NfsB-mCherry) background, Tg (4XUAS: NLS-Cas9, cmlc2: RFP) is Tg (mpx: KALTA4 / UAS: NfsB-mCherry) and Tg (pax7b: Gal4; UAS: GFP) backgrounds. Co-segregation of the three transgenes by F0 and F1 backcrossing to this background was performed by selecting embryos with appropriate fluorescence [Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry/4XUAS:NLS- CAS9, cryaa:EGFP): red macrophages confirmed the presence of the mpeg1:GAL4FF transgene and green eyes sorted to confirm the presence of the cryaa:EGFP marker gene linked to 4xUAS:NLS-Cas9 (this The strain is called mpeg1-Cas9). Tg (mpx:KALTA4/UAS:NfsB-mCherry/4XUAS:NLS-Cas9, cmlc2:RFP): Red neutrophils and red hearts were sorted (this line is called mpx-Cas9). Tg(pax7b:Gal4; UAS:GFP/4XUAS:NLS-Cas9, cmlc2:RFP): green muscle stem/progenitor cells and red heart were sorted (this lineage is called pax7b-Cas9)]. For gene editing experiments, embryos incross from adults of the F2 or F3 generation were used. Targeting nampta and ccr5 used the gRNA or double gRNA combination used to generate the mutants (see above). Dual gRNA combinations were used for targeting of nmptb. These gRNAs were generated using namtb crRNA_1,5'-ttctctgaccaaaacgcaAGG-3' and namptb crRNA_2,5'-gttgacctgtgaacgtgataGGG-3'. The efficiency of individual gRNAs of nampta, double gRNAs of ccr5 and namptb, was tested in whole embryo gene editing and was shown to have a transformation efficiency of 89-100%. For tissue-specific gene editing, 3 nL gRNA mix [3 μM gRNA (1.5 μL of each gRNA if dual gRNAs are used) + 0.5 μL 2% phenol red + 1.5 μL 0.1 M KCl mix ] were injected into the cells of 1-cell stage embryos.

Visualization and Quantification of NAD/NADH In vivo two-photon excited fluorescence of NADH in zebrafish larvae was measured with an Olympus FVMPE-RS upright multiphoton microscope using a 25X/1.05 water immersion objective. A wavelength of 810 nm and a 450/70 bandpass filter were used. The same wavelength and 610/70 bandpass filter was used for detection of mCherry fluorescence. A galvo scanner was used to generate high-resolution datasets, and an 8 kHz resonance scanner was used for time-lapse imaging to minimize phototoxic effects.

In vitro total NAD ⁺ and NADH levels and their individual levels (to determine their ratios) were measured using the NAD/NADH-Glo™ assay (Promega) according to the supplier's instructions. Measurement of total NAD/NADH: Assays were performed on macrophages sorted from macrophage-specific nampt knockout larvae (mpeg1-Cas9+nampt gRNA injected) and control (mpeg1-Cas9) larvae. Control larvae at 2 dpf were immersed in either the NAMPT enzyme inhibitor GMX1778 (10 μM) (Sigma-Aldrich) or nicotinamide mononucleotide (NMN) (100 μM), the rate-limiting enzymatic catalytic product of NAMPT (Sigma-Aldrich). . These were used as additional controls to confirm the detection range of the assay. 3 dpf of mpeg1 ⁺ macrophages were sorted from each group at a density of 2000 cells/well into white flat-bottomed 96-well plates (Costar) containing 50 μL PBS. Cells were incubated with 50 μL of NAD/NADH Glo™ detection reagent. After incubation for 1 hour, luminescence was measured with a microplate reader (BMG PHERAstar; gain 3600, integration time 1 second). Each measurement point represents the average luminescence response measured in relative luminescence units (RLU). Separate measurements of NAD+ and NADH: Tg (mpeg: GAL4FF/UAS: NfsB-mCherry) isolated from 4 dpf intact larval trunks and 1 dpi (5 dpf) and 2 dpi (6 dpf) muscularis needlestick injury larval sites. Assayed against FACS-sorted mCherry ⁺ macrophages. Cells were sorted at a density of 5000 cells/well into white flat-bottomed 96-well plates (Costar) containing 50 μL PBS. Two separate reactions were performed to remove either NAD ⁺ or NADH according to the manufacturer's instructions, and the remaining metabolites were detected with 100 μL of NAD/NADH Glo™ detection reagent. After incubation for 2 hours, luminescence was measured in the same manner as described above, and the NAD ⁺ /NADH ratio was calculated from the amounts of NAD ⁺ and NADH.

RT-PCR
Total RNA was extracted from pax3a ⁺ cells sorted by FACS using TRIzol™ Reagent (Thermo Fisher). cDNA was synthesized using the iScript™ Advanced cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instructions. For RT-PCR, 10 μl reactions were set up using GoTaq® Green Master Mix (Promega). The primers used for amplification of zfccr5 are 5'-TTATAACCAAGACATGTCGGCG-3' and 5'-ACCCAGACGACCAGACCATT-3'. Primer pairs designed to span a 388 bp intron such that the cDNA and genomic DNA (gDNA) templates yielded bands of 191 bp and 579 bp, respectively. The cycling protocol was performed as follows: 25 cycles of initial denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 58°C for 1 minute, extension at 72°C for 45 seconds, and a final 72 cycles. C. for 5 minutes. Since this cycle can amplify both cDNA and gDNA, the presence or absence of genomic DNA contamination can be confirmed.

Total RNA was extracted from 4dpf zebrafish larval tails of different genotypes and cDNA was synthesized (mutant, heterozygous, homozygous siblings were larval heads from nampta ^+/pc41 ccr5 ^+/pc42 crosses). (identified by partial genotyping). RT-PCR was performed using the following primer pairs. These are all designed to span an intron; '-GAGGGAAAATTAAGCTCAGAAGG-3' (wild-type amplicon 657 bp), zfactc1b_housekeeping_RT: 5'-TTGACAACGCTCCGTATG-3' and 5'-GCCAACCATCACTCCCTGAT-3' (amplicon 110 bp).

Maximum Likelihood Phylogenetic Tree Analysis Putative Ccr5 orthologues in zebrafish were identified by BLAST and orthology assessed by phylogenetic analysis. Amino acid sequences were aligned with MUSCLE and trimmed with GBLOCKS. Using PHYML, trees were created using maximum likelihood methods using the JTT model of protein evolution (estimated with ProTest v3.4.2). Trees were visualized using iTOL (v4.3) (70-76).

Statistical Analysis Embryos were randomly assigned to each treatment group and the experimenters were blinded to the groups prior to analysis. All experiments were performed in at least three independent biological replicates and exact numbers are shown in the figures. Statistical analysis was performed using Prism version 7.0c (GraphPad Software, Inc.). Data were analyzed using Student's unpaired two-tailed t-test when comparing two conditions and Tukey's analysis of variance (ANOVA) with post-hoc analysis when comparing multiple conditions.

Production and purification of NAMPT402-491 Final protein concentration: 280 μg/mL
Expected size: 11 kDa
Observed size on gel: 14-15 kDa (data not shown)
12% SDS-PAGE:
Lane 1 NAMPT402-491
Lane 2 MW marker (kDa)
Amino acid sequence:
MSYVVTNGLG VNVFKDPVAD PNKRSKKGRL SLHRTPAGNF VTLEEGKGDL EEYGHDLLHT VFKNGKVTKS YSFDEVRKNA QLNIEQDVAP HHHHHHH
Number of amino acids: 97 (including SeptaHis C-terminal part in this example)
Molecular weight: 10977.26
Extinction coefficient (used for calculation:
This protein does not contain Trp residues. Experience shows that the calculated extinction coefficients can be in error by 10% or more.
The unit of extinction coefficient is M-1 cm-1, wavelength 280 nm, measured value in water.
Extinction coefficient 4470
Abs 0.1% (=1 g/l) 0.407 (value used for reading in Nanodrop method)

Preparation protocol Plasmid preparation and bacterial transformation:
- The NAMPT402-491 DNA sequence was subcloned into the pET22b(+) vector (plasmid map here).
- Vector transformed into competent E. coli (strain BL21DE3) by heat shock at 42°C for 30 seconds.
- The transformed bacteria were plated on ampicillin/agar plates and single colonies were selected.

Protein production:
- Overnight culture with 5 mL starter culture, LB + 100 μg/mL ampicillin.
- After incubation, the starter culture was diluted 1:100 with LB + 100 μg/mL ampicillin and expanded to an OD600 of 0.7 (~4 hours).
- Induction with 1 mM IPTG for 4 hours.
- The bacterial culture was spun at 4000 g for 20 minutes and the pellet was resuspended in lysis buffer (PBS + 50 mg Lysozyme + 1% Triton X-100 + 2.5 U Benzonase + 10 mM MgCl2 + 1 tablet of Complete EDTA-free protease inhibitor).
- 4 x 30 sec sonication and 30 min incubation at 4°C.
- The lysate was spun at 14000 g for 30 minutes and the supernatant was filtered prior to protein purification.

Protein purification - Load lysate onto HisTap HP (GE healthcare).
- LPS was removed by washing with Triton X-114 0.1%.
- 2mM ATP + 10mM MgCl2 wash to remove bacterial chaperones.
- 20mM imidazole wash to remove other contaminants.
- Protein elution with imidazole gradient from 20 mM to 500 mM.
- Pool fractions containing pure protein and dialyze against PBS overnight.
- Concentrate proteins using 3kDa amicon filters.

This description is further illustrated by the following examples, which should not be construed as limiting in any way.

Live imaging of cellular responses to tissue injury remains a long-term goal of the regenerative medicine field. In an attempt to reach this goal, we used a previously developed zebrafish model of tissue injury in which the optical accessibility of larvae allows the application of non-invasive techniques to assay repair in real time [Gurevich D. B. et al. Asymmetric division of clonal muscle stem cells co-ordinate muscle regeneration in vivo. Science (2016)] (3).

Here, we apply this approach to regenerating skeletal muscle with the aim of clarifying the cellular and molecular events that define regeneration in vivo.

By subjecting a transgenic zebrafish reporter strain in which cellular components present in the wound site to fluorescent labeling to acute injury, the location and response kinetics of cells occupying individual wound sites are correlated with the stem cell compartment during muscle repair. became possible.

Two injury paradigms were employed: a focal laser ablation model that cuts 2-6 muscle fibers in a single injury, and a needle stick model that consistently injured two adjacent sarcomeres. Both models systematically analyzed differential cellular behavior within the regenerative niche, revealing a directional and obligatory association between specific macrophage populations and muscle stem cells during regeneration. Different modalities of high-resolution imaging and in vivo cell tracking revealed these interactions with high temporal and spatial resolution.

Priming Specific Macrophage Subsets to Respond to Trauma First, we used multiphoton imaging to capture responses before and after laser ablation muscle injury. Combined transgenic line Tg(actc1b:GFP);Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), in which differentiated muscle expresses green fluorescent protein (GFP) and macrophages are labeled with mCherry fluorescence; Observing the dynamics of responsive macrophages, we were able to define the wound area [Fig. 1a-c, Supplementary Video 1 (not shown)]. After injury by laser ablation of muscle fibers, 34±2% of macrophages (MΦ) near the wound site (within a radius of 260 μm from the center of laser ablation, areas encompassing muscle layers 2 on both sides of the injury, n=4 wounds) , showed rapid, brisk, directional migration towards the wound site , Fig. 1c, Supplementary Video 1 (not shown)]. The fact that not all macrophages near the lesion responded to lesions suggested the possible existence of specific macrophage subsets primed to respond to trauma.

Next, we investigated the dynamics of migrating macrophages at the boundary of the wound site after muscle fiber laser ablation with cellular resolution. High-resolution serial confocal imaging showed that the number of macrophages within the wound site peaked at 2.50±0.42 hours post-injury (hpi) (n=8 injury), after which additional macrophages entered the wound site. It was shown not to migrate [Fig. 1d-f, Supplementary Video 2 (not shown)].

Of all injury-responsive macrophages, 51.11±1.83% (n=8 injuries) remained within the wound site up to 24 hpi (FIG. 1e-f). Macrophages located at these long-term injury sites are termed "resident." On the other hand, macrophages exiting the wound site prior to 10.48±1.19 hpi were termed 'transient' (Fig. 1d,f).

High-resolution confocal imaging reveals that these two macrophage populations exhibit morphological differences, with transient cells exhibiting a distinct astrocytic appearance, whereas resident cells can adopt a more spherical morphology. It was found [residual MΦ showed 0.248±0.022 higher sphericity values compared to transient MΦ, Fig. 1g-i, Supplementary Video 3 (not shown)]. This transition of transient to resident macrophages occurred regardless of the size of the injury, but was temporally proportional to the size of the injury [51.6% of the needlestick injury-responsive MΦ transitioned to resident status by 2 days later (dpi), n=20, Figures 5a-d].

The phylogenetic relationships between different subsets of wound-active macrophages remain controversial in the field. To clarify whether resident macrophages are a subset of the original pool of injury-responsive macrophages or the result of the migration of another macrophage, we labeled macrophages with the photoactivatable fluorescent protein Kaede Tg(mpeg1:Gal4FF /UAS: Kaede) transgenic lines were utilized. One day after needlestick injury, transient macrophages present within the wound site were photoactivated and distinguished from macrophages outside the injury (n=20, Figures 1j-k''). When the injured site was re-imaged 1 day later, all resident macrophages within the wound site showed photoelectric conversion of kaede (FIGS. 111-m). This indicates that resident macrophages are a subset derived from the initial transient injury-responsive macrophage population and are not the result of separate migrations that differ in time.

The respective contributions of tissue-resident and recruited macrophages in the muscle repair process are an area of further active research. Combining light-sheet technology with zebrafish larval models provides a unique opportunity to explore this field, as it provides a whole-organism perspective on the movement of individual macrophages during the repair process. Thus, individual macrophages were exposed to light throughout zebrafish larvae from 10 min to 25 hpi after laser ablation muscle fiber injury of Tg(actc1b:GFP) and Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry) transgenic lines. Tracked by sheet microscopy [Supplementary Video 4 (not shown)]. Retrospective tracking of macrophages located at the injury site showed that all macrophages that adopted a resident phenotype migrated from the vicinity of the injury site and thus became tissue-resident rather than migratory. (n=4 lesions, Figure 1n-n'). These observations suggested that there are local anatomical constraints on the activation of wound-responsive macrophages in zebrafish larvae. This localized response exemplifies the previously reported bioprotective 'shielding' function of tissue-resident macrophages (isolating microlesions in various tissues, including muscle, and preventing extraneous neutrophil migration). It was evocative. However, in the context of muscle injury, it remains unresolved why macrophages in close proximity to the wound refrain from migrating to the injury site.

Although macrophages are essential for efficient skeletal muscle regeneration, a resident macrophage population is also clearly required for injury repair. Therefore, we next compared muscle regeneration with and without transient and resident macrophage populations to determine whether they were required for this process. A Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry) transgenic line can timely and transiently control nitroreductase-mediated gene disruption of macrophages by the addition of metronidazole (Mtz) at discrete time points during muscle regeneration. [Fig. 2a, Fig. 5e-f, Supplementary Video 5 (data not shown)]. Addition of Mtz in this transgenic line resulted in efficient cell death and ablation of macrophages was confirmed after 3 hours of treatment. After 10-13 hours, most of the macrophages present in the trunk were ablated and removed from the larva by their cell death (Fig. 5g, Supplementary Video 5). Moreover, this ablation strategy did not alter other innate immune cell responses to muscle wounds at 2 dpi (Fig. 5h-i). Regeneration after needle-prick muscle injury was assessed by birefringent imaging of the wound site at key junctures during the course of the repair process (n=24 per group, Figures 2b-h). The pseudocrystalline arrangement of muscle sarcomeres confers an intrinsic birefringence and when viewed with polarized light, undamaged muscle appears bright against a black background, allowing non-invasive visualization of muscle integrity [Berger et al. , et al. (2012)]. Addition of Mtz at the time of injury (to ablate all injury-responsive macrophages) and Mtz treatment at 1.75 dpi (to preferentially ablate resident macrophages) both resulted in marked regeneration defects (Fig. 2b-h, Fig. 5g'). To demonstrate that the observed repair defects were solely due to macrophages and not the result of toxicity by Mtz or a response to general innate immune deficiencies, the same strategy used to ablate macrophages was used. A previously characterized Tg (mpx:KALTA4/UAS:NfsB-mCherry) transgenic line that allows genetic ablation of neutrophils by nitroreductase was used. Neutrophils are another important cellular component of the innate immune response of larval zebrafish to wounds, and Mtz treatment at the time of injury aimed at ablating all neutrophils responding to injury. , did not inhibit muscle regeneration (Fig. 5j-o). Thus, macrophages are not only essential for efficient skeletal muscle regeneration, but a resident macrophage subpopulation is clearly required for wound repair.

The defective repair seen in macrophage-deficient cells is not due to poor clearance of debris, but rather to an unexplained lack of promyogenic function.
Wound debris removal is performed by many types of cells, most of which are due to phagocytic macrophages. One possible explanation for the lack of regeneration observed when macrophage populations are killed early in the regeneration process is lack of clearance or no effect. Four separate approaches were taken to assess the clearance of wound debris when all macrophages responding to injury had died. First, larvae were stained with fluorescently-conjugated phalloidin to detect the actin cytoskeleton of necrotic skeletal muscle fibers remaining at the injury site (Fig. 6a). Second, a Tg (ubi:secAnnexinV-mVenus) transgenic line was utilized to identify apoptotic cells within the lesion (Fig. 6b-c). Third, LysoTracker fluorescent vital dyes were used to visualize acidic membrane organelles and autophagic processes (Fig. 6d-e), and finally, hematoxylin and eosin staining was performed to remove dead fibers at the wound site. was monitored (Fig. 6f). No accumulation of cellular debris at the injury site in macrophage ablated larvae compared to non-ablated controls was observed in any of these independent approaches (Fig. 6a-f). These data suggest that the poor repair of macrophage-deficient cells is not due to poor debris clearance, but to an unexplained lack of promyogenic function.

Macrophages maintain highly dynamic membrane contacts with stem cells, enveloping them in continuous and repetitive membrane expansions, ie macrophage-stem cell association may be essential for stem cell proliferation.
Since the positions occupied by resident macrophages within the repair niche are topographically similar to those identified as containing dividing muscle stem progenitors (Fig. 1e), we next hypothesized that these We investigated the spatial and temporal relationships of the two cell populations. Long-term confocal time-series imaging after muscle injury by laser ablation was performed using a combined Tg (mpeg1: GAL4FF/UAS: NfsB-mCherry); was performed in a genetic strain [Fig. 2i-l, Supplementary Video 6 (data not shown)]. The pax3a:GFP reporter is particularly useful in this context because it is expressed in both the muscle stem cell compartment and the dividing progenitor population. After injury, both transient macrophages and pax3a ⁺ cells responded and co-migrated to the wound site. However, pax3a ⁺ cells migrated independently of transient macrophages, exhibited different migration kinetics, and were resident in the niche at the wound edge by 10.25±1.99 hpi (n=5 injury, 2i-l, 7d-f, Supplementary Video 6, not shown). After the transition from the transient macrophage phenotype to the resident macrophage phenotype, resident macrophages associated with pax3a ⁺ cells covering the wound edge at 11.17 ± 1.13 hpi and 5.38 ± 1.79 h. It continuously interacted with neighboring stem cells over the course of [Fig. 2l-m, Supplementary Video 6 (data not shown)]. These interactions differ in their nature and duration when compared to the short-term interactions between transient macrophages and pax3a ⁺ cells, which are maintained for an average of 12.86 ± 11.95 min (n = 37 interactions, Figures 2i-k, m). This set of interactions is the Tg (met: mCherry-2A-KALTA4/UAS: NfsB-mCherry) muscle stem cell marking line ³ and GAL4FF integrated into the pax7b gene and used to drive Tg (UAS: NfsB-mCherry). was also reproduced in analyzes using the [Tg(pax7b:GAL4FF)] myogenic stem/progenitor marker gene trap line ¹¹ (Fig. 7a-c, Supplementary Video 9).

The direct role of specific macrophage subsets in the control of muscle regeneration To better visualize these cell-cell interactions, high-resolution live imaging using AiryScan confocal microscopy was performed on resident macrophages and pax3a ⁺ muscle stems. / progenitor cells [Fig. 2n, Supplementary Video 7 (data not shown)]. A persistent association was observed between resident macrophages and stem cells. Macrophages maintained highly dynamic membrane contacts with stem cells, wrapping them in continuous and repetitive membrane extensions [n = 10 lesions, Fig. 2n, Fig. 7g, Supplementary Videos 7, 8 (data shown). figure)]. After these prolonged physical interactions, the assembled pax3a ⁺ muscle stem cells invariably underwent cell division [Fig. 2n, Supplementary Videos 7, 8 (data not shown)]. These macrophage-stem cell interactions were further validated by correlated light and electron microscopy (CLEM), which confirmed that the two cell types exhibited tight membrane attachment in the 3D XYZ plane (Fig. 8a-8). d). After these prolonged physical interactions, the associated pax3a+ muscle stem cells invariably underwent cell division (Fig. 2n, Fig. 7g, Supplementary Movies 7, 8). On average, resident macrophages interacted with a particular stem/progenitor cell for 5.42±1.72 hours (n=10 interactions) before the associated stem cells divided. This is comparable to the time a subset of resident macrophages maintain uninterrupted interaction with wound-responsive muscle stem cells (5.38±1.79 h, Fig. 2l-m), indicating that resident macrophages once It was emphasized that once interacting with stem cells, it ceases with stem cell division. Importantly, in the absence of long-term interaction with resident macrophages, no muscle stem cell division was observed (n=26 wound-associated stem cell divisions, n=10 independent larval ). 71% of the proliferation-induced macrophages maintained long-term interactions with a single muscle stem cell, 29% interacted with two stem cells simultaneously, and interacted with stem cells for two or more simultaneous ground times before cell division. No macrophages remained active (n=9 stem cell divisions). After stem cell proliferation, macrophages and daughter myoblasts migrated away from each other, and macrophages became localized in the muscular septum 1.94±0.94 hours after stem cell division. During this migration, 44% of macrophages associated with one or both daughter myoblasts for 1.45±0.69 hours, the rest did not do so (n=9 stem cell divisions, Figure 7h, supplementary video 10). Interestingly, the nature of these interactions between muscle stem cells and wound-coating macrophages phenotypically dictates the dendritic cell-T cell immunological synapse within the lymph node leading to T cell activation and proliferation. is reminiscent of Taken together, these data suggest that binding between macrophages and stem cells may be essential for stem cell proliferation.

To investigate resident macrophages required for stimulation of stem cell proliferation, EdU labeling of needle-prick muscle-injured larvae from which resident macrophages were ablated was performed (Fig. 2o-q)). At the wound site of 2 dpi control larvae, all EdU ⁺ proliferating cells corresponded to pax3a ⁺ myogenic stem/progenitor cells. Ablation of resident macrophages markedly reduced the proliferation of pax3a ⁺ muscle stem cells within the injury site and reduced the number of EdU-positive cells to the constitutive levels present in non-injured areas (pax3a ⁺ muscle stem cell proliferation 51% reduction in , n=13, Fig. 2p-q). These results reveal a surprisingly direct role for specific macrophage subsets in regulating muscle regeneration, and that a fraction of wound-attracted macrophages form a transient stem cell niche. have demonstrated Ablation of this niche-specific macrophage subset markedly reduced the number of myogenic precursors present at the injury site, resulting in impaired muscle regeneration (Fig. 2f-h).

Unsupervised clustering identified a homogeneous population (cluster 3) from unwounded tissue, cluster 2 of transient macrophages, and many discrete clusters of wound-specific resident macrophage subtypes.
To characterize the injury-responsive macrophage population, single-cell RNA sequencing (scRNA-seq) was performed on injury-site macrophages isolated from discrete stages of the regeneration process. After needlestick skeletal muscle injury of Tg(mpeg1:mCherry) larvae, the wound area was dissected at 1, 2, 3 dpi and mCherry-expressing macrophages were sorted by FACS (Fig. 3a). Macrophages sorted from intact larvae were also included in the analysis. Unsupervised clustering identified seven distinct clusters of macrophage subtypes (Fig. 3b). Of note, cells in all clusters expressed the pan-leukocyte marker L-plastin (lymphocyte cytoplasmic protein 1, Icp1) and the pan-macrophage marker cd163, verifying their macrophage identity (Fig. 3e, Fig. 9a, p Table 1). Unsupervised clustering identified eight distinct clusters of macrophage subtypes (clusters 0-7) (Fig. 3b). This analysis revealed the presence of greater macrophage heterogeneity than previously reported during muscle or other ^regeneration scenarios (Fig. 3b). We then compared the time points at which the injury-responsive macrophages were isolated and the identified clusters overlaid on the UMAP scattergram to determine whether the time of injury corresponded to specific individual clusters (Fig. 3c). Intact larval macrophages formed temporally uniform clusters (cluster 3, Fig. 3d, Supplementary Table 2). Moreover, specific unsupervised clustering of macrophages from intact larvae did not reveal any additional complexity, reconfirming their homogeneity. The lack of such pre-determination in the intact macrophage population is interesting given that only a fraction of muscle-resident macrophages migrate to and respond to injury. This suggests that the ability to respond to wound-derived cues is an acquired condition, at least at the level of gene expression. The majority of transient macrophages (1 dpi) were also clustered (cluster 1, Fig. 3d, Supplementary Table 2), suggesting that macrophages migrating to the lesion are systematically initially activated. The remaining six clusters were composed of resident macrophages (2-3 dpi), highlighting their heterogeneity (clusters 0, 2, 4, 5, 6 and 7, Fig. 3d, Supplementary Table 2). Next, we performed transcriptome trajectories and pseudo-temporal analyzes to better understand the potential phylogenetic relationships among the identified resident macrophage clusters. These analyzes revealed that there are two 'mature resident' macrophage subtypes, represented by clusters 2 and 6. All other clusters possess properties suggestive of transitional subtypes from which these mature subsets arise (Fig. 3g-k).

In mammals, activated macrophages have been defined by in vitro studies as a simple dichotomization of alternatingly activated anti-inflammatory macrophage pools (M2 ²⁰ ) with inflammatory macrophages (M1). A recent analysis revealed that this simple dichotomy of macrophage phenotypes does not represent macrophage diversity in vivo, although specific markers were used to distinguish between inflammatory and anti-inflammatory subtypes. It is possible to identify Analysis of differentially expressed genes between each cluster indicates that cluster 2 macrophages have mammalian anti-inflammatory properties, including arginase 2 (arg2), matrix metalloprotease 9 (mmp9) and matrix metalloprotease 13 (mmp13) ^21-23 . It was found to be particularly enriched in subtype markers (53.4%, 100%, 98.6% of cells residing in cluster 2 express arg2, mmp9 and mmp13, respectively, Fig. 3e, Supplementary Table 3).

Since all cluster 2 cells expressed mmp9 (Fig. 3e,k), in situ hybridization to endogenous mRNA (Fig. 3l,o) and transgenic lines expressing EGFP under the control of the mmp9 promoter (TgBAC (mmp9:EGFP) ²⁴ , Fig. 3m-n, Fig. 10a and Supplementary Video 11) were used to assess spatiotemporal expression after injury. These analyzes revealed that mmp9 expression was upregulated after 2 dpi in the resident macrophage population associated with muscle stem cells present at the wound site (FIGS. 31-m, o). Quantitative analysis confirmed that at 2 dpi, 71.09±12.05% (n=11) of mpeg ⁺ resident macrophages at the wound site were mmp9 ⁺ (Fig. 3m–o), with a range of , consistent with scRNA-seq analysis that confirmed that 59% of all 2dpi macrophages express mmp9 (Fig. 3f). The number of mmp9 ⁺ macrophages is also consistent with the number of resident macrophages present at the wound site that become actively interacting with muscle stem cells (72.92±20.83%). Furthermore, the majority of resident macrophages remaining at the wound site at 2 dpi expressed markers specific for cluster 6 (15.10±12.13% of cells were Pou2f3 ⁺ /mpeg ⁺ , cells 23.36±12.85% of the Prox1a ⁺ /mpeg ⁺ , Fig. 9b-g). This observation is strongly in line with the scRNA-seq trajectory and pseudotemporal analyzes described above (Fig. 3g-k), which define clusters 2 and 6 as two 'mature resident' macrophage subtypes present during late wound regeneration. Correlated. Furthermore, in contrast to cluster 2 cells, the majority of cluster 6 cells are not located adjacent to pax3a ⁺ muscle stem/progenitor cells [at 2 dpi, Pou2f3 ⁺ /mpeg ⁺ cells (n=8) 12 ± 10% of and 9 ± 7% of Prox1a ⁺ /mpeg ⁺ cells (n = 10) were adjacent to pax3a ⁺ cells present at the wound site]; It is emphasized that it does not actively interact with stem cells.

To more directly engage cluster 2 macrophages in the regulation of regenerative muscle stem cell proliferation, an alternative mmp9 promoter-driven transgenic line [TgBAC(mmp9:EGFP-NTR) ²⁴ ] expressing a nitroreductase enzyme fused to EGFP was developed. was used to specifically ablate these cells during the repair process. These larvae underwent a needlestick injury at 4 dpf (0 dpi). When 1.75 dpi larvae were treated with 5 mM Mtz, this administration specifically ablated 77.94% of the mmp9 ⁺ /mpeg ⁺ macrophages present at the lesion (Fig. 10b-d). Specific ablation of these cells markedly reduced skeletal muscle repair (Fig. 3p–q) and markedly reduced muscle stem cell proliferation, as demonstrated by the ablation of mpeg ⁺ resident macrophages described above. (Fig. 2f-h, Fig. 3r, Fig. 10e-g). Taken together, these analyzes indicate that cluster 2 has a resident macrophage subset associated with specific mmp9 ⁺ muscle stem cells that is required for muscle stem cell proliferation during muscle regeneration.

Cluster 4 (reclassified to cluster 2) macrophages are specific resident macrophages that express arg2, mmp9 and mmp13 and associate with muscle stem cells. Cells of cluster 4 (reclassified to cluster 2) are enriched for mammalian M2 markers such as arginase 2 (arg2), matrix metalloprotease 9 (mmp9), matrix metalloprotease 13 (mmp13) and thus macrophage-specific anti-inflammatory Sexual subtypes appeared to be recurrent (53.5%, 100%, 98.6% of cells present in cluster 4 express arg2, mmp9 and mmp13, respectively). Since all cluster 4 (reclassified to cluster 2) cells expressed mmp9, its spatiotemporal expression was measured after injury, showing spatial restriction at the wound site and resident muscle stem cell-associated cells. It was upregulated after 2 dpi in the macrophage population (Fig. 3f-h). These analyzes show that cluster 4 (reclassified as cluster 2) contains a resident macrophage subset associated with specific muscle stem cells.

Note that the cluster numbers of the identified macrophage clusters were renumbered as follows: previous version cluster 3 (intact) → current version cluster 3, previous version cluster 4 (mmp9 ⁺ ) → present. version cluster 2, previous version cluster 6 (pou2f3 ⁺ /prox1a ⁺ )→current version cluster 6. By characterizing these macrophage clusters (Cluster 1-8) as shown in the Supplementary Table, selection and/or enrichment of macrophage clusters can be performed, for example, by functional characterization based on differences in their gene marker expression profiles, as well as in vitro and in vivo behavior, such as anti-inflammatory, differentiation-promoting activity.

The NAMPT-CCR5 signaling axis induces muscle stem cell proliferation in vivo. A list of genes encoding secreted pro-mitogenic molecules that were specifically upregulated within cluster 4 (reclassified as cluster 2) compared to all other identified clusters [Extended Data Table. 1 (data not shown)] was evaluated. This is consistent with a dataset consisting of receptors known to be expressed on muscle stem/progenitor cells [Yahiaoui, L., Gvozdic, D., Danialou, G., Mack, M. & Petrof, B. J. CC family chemokines directly regulates myoblast responses to skeletal muscle injury. The Journal of Physiology 586, 3991-4004 (2008)] (21), thereby regulating nicotinamide phosphoribosyltransferase (NAMPT/Visfatin/PBEF) and the chemokine receptor CCR5. (Fig. 3e). NAMPT is a multifunctional protein that, when localized intracellularly, plays well-known roles in cell metabolism and NAD regeneration, but its role as a secreted factor is less well-known. Its secreted form, secretory NAMPT (secNAMPT), functions as a cytokine and has been reported to have physiological and pathological functions [Grolla, A. A., Travelli, C., Genazzani, A. A. & Sethi, J. K. Extracellular nicotinamide phosphoribosyltransferase, a new cancer metabokine. British journal of pharmacology 173, 2182-2194 (2016)(25)]. In contrast to their intracellular functions, information on the role of secreted proteins is conflicting and poorly understood, not only in terms of mode of secretion but also in terms of mechanism of action. Despite this lack of mechanistic insight, secNAMPT has been demonstrated to be promitogenic and reported as a regulator of a wide range of regenerative potential in different tissues [Grolla et al. (2016)]. CCR5 has been suggested as a putative cell surface receptor for NAMPT [Van den Bergh, R. et al. Monocytes contribute to differential immune pressure on R5 versus X4 HIV through the adipocytokine visfatin/NAMPT. PloS One, e35074 (2012) (26)]. Furthermore, NAMPT has been demonstrated in vitro to alter the expression of myogenic regulatory factors in myoblasts. As a result, we hypothesized that the NAMPT-CCR5 signaling axis promotes muscle stem cell proliferation in vivo.

To investigate whether the NAMPT/CCR5 signaling axis controls muscle stem cell-mediated repair in vivo, we investigated two nampt genes encoded in the zebrafish genome: nampta (a homolog of a single Nampt-encoding gene in mammals, clustered together). The spatiotemporal expression of ³² in wound-resident macrophages in vivo Within, a number of independent approaches were used to measure expression. First, we performed in situ hybridization analysis after larval muscle injury and found that nampta was specifically upregulated at the wound site at 2 and 3 dpi (Fig. 10h), whereas namptb was at a constant low level from 1-3 dpi in the injury area. (Fig. 13k). These observations are consistent with the scRNA-seq data that identified nampta as the only upregulated gene in cluster 2 (Fig. 3e, Supplementary Table 3). Next, we identified a NAMPT antibody that recapitulated the endogenous developmental expression profile of ^nampta32 (Fig. 10k), and based on the results, combined Tg (mpeg1: GAL4FF/UAS: NfsB-mCherry); Antibody staining was performed on the genetic lines. These analyzes revealed that expression of Nampt at the injury site was temporally upregulated at 2 dpi (Fig. 10l–o), indicating that Nampt expression at the injury site was associated with stem cell-associated resident macrophages as well as cells at the injury site. It was also confirmed to be specifically localized in the outer space (Fig. 4a). Of the resident macrophages present at the lesion site at 2 dpi, 72±16% (n=8 lesions) were consistent with scRNA-seq analysis that identified 58% of 2 dpi macrophages expressing Nampt ⁺ /mpeg1 ⁺ (nampta). value range, Fig. 3f). This quantification is based on the percentage of resident macrophages (72.92±20.83%) that come to interact with muscle stem cells, and the percentage of mpeg ⁺ macrophages that also express mmp9 ⁺ at the 2 dpi wound site (71. 09±12.05%). All Nampt ⁺ /mpeg1 ⁺ double positive cells were associated with pax3a ⁺ muscle stem/progenitor cells (Fig. 4a). Furthermore, full-thickness immunohistochemical analysis of regenerating larvae showed decreased Nampt levels after ablation of macrophages, but not neutrophil-specific ablation, again confirming that the source of secreted Nampt within muscle injury is from macrophages. (Fig. 10n-o).

To monitor the level and cause of increased NAMPT activity in muscle regeneration in vivo, we exploited the critical physiological role of intracellular NAMPT in catalyzing the rate-limiting process of the NAD salvage pathway. It is known that when the activity of NAMPT increases, the level of intracellular NADH increases. NADH exhibits intrinsic fluorescence with an excitation peak at 365 nm and can be observed with a two-photon excitation fluorescence microscope. We therefore developed an assay to examine NADH levels in vivo and used it as a proxy to estimate intracellular Nampt levels in real time under the wound environment [Hong, SM et al. Colon cancer progression by suppressing reactive oxygen species level. Cancer science 110, 629-638 (2019)]. At 2 dpi, only resident macrophages specifically expressed high levels of NADH within the wound site (Fig. 10i, Supplementary Movie 12), and using a luminescence-based assay, NAD within isolated wound-site macrophages ⁺ and NADH levels could be quantified, confirming the in vivo-based observations (resident MΦ had a 0.6781±0.1650 lower NAD ⁺ /NADH ratio compared to transient MΦ, FIG. 10j). These findings confirm that the increased Nampt production observed at regenerating wound sites occurs specifically within resident macrophages, highlighting its role as a major source of Nampt at the wound site. is. Furthermore, the high levels of NADH in resident macrophages and the resulting reduction in the NAD ⁺ /NADH ratio may be due to a metabolic shift, particularly in the cluster 2/mmp9 ⁺ resident macrophage subtype. There is growing evidence that the metabolic state of macrophages is associated with their activation [Watanabe, R. et al. Glucose metabolism controls disease-specific signatures of macrophage effector functions. JCI insight 3 (2018)]; Analysis of our scRNA-seq dataset revealed a potential glycolytic shift in the mmp9 ⁺ subset (Fig. 9h-i). Alternatively, high Nampta expression levels may occur because intracellular stores of Nampta are secreted and depleted at sufficient levels, resulting in the induction of high levels of Nampta expression in resident macrophages. There is also

Induction of myoblast proliferation requires selective signaling of NAMPT through the CCR5 receptor. To better elucidate the molecular basis of NAMPT function in muscle stem cells, an in vitro assay with mammalian cell culture was performed. The production of molecular reagents and recombinant proteins in mammalian systems has enabled rigorous evaluation of stem cell activation models generated from in vivo observations of zebrafish. First, we aimed to determine whether secNAMPT acts through its putative receptor, CCR5, to regulate muscle stem cell proliferation. Since there is only one report that NAMPT and CCR5 interact directly, we decided to evaluate the binding of NAMPT and CCR5 independently. The binding of human recombinant NAMPT [hrNAMPT source 1 (hrNAMPT ₍₁₎ )] to human recombinant CCR5 receptor (hrCCR5) was assessed by an enzyme-linked immunosorbent assay (ELISA) approach. As a result, hrNAMPT ₍₁₎ bound to hrCCR5 with a dissociation constant (K _D ) of 172±18 nM (Fig. 11a), confirming previous findings ³⁰ . NAMPT is a highly evolutionarily conserved protein, with human NAMPT sharing 96% and 88.24% identity with mouse NAMPT and zebrafish Nampta proteins, respectively. Since we have utilized hrNAMPT in the murine cell line assays described below, the affinity of hrNAMPT for murine recombinant CCR5 (mrCCR5) was also assayed by a competitive ligand binding assay with its cognate ligand, CCL4. Here, mouse recombinant CCL4 (mrCCL4) exhibited a half-maximal inhibitory concentration ( _IC50 ) of 34.4±2.2 nM, demonstrating the strong affinity of hrNAMPT for mrCCR5 and binding to the receptor through the same site as CCL4. (Fig. 11b).

We next investigated whether NAMPT initiates a growth-promoting signaling cascade on muscle stem cells using an in vitro mouse cell culture model to assess whether the NAMPT-CCR5 interaction functionally regulates this process. . For this, we utilized mouse C2C12 myoblasts. These myoblasts do not appear to be actively secreting NAMPT, as NAMPT is either undetectable or very low detected in the C2C12 secretome data. However, these cells have previously reported physiologically relevant levels of CCR5 [Gilliam, BL, Riedel, DJ & Redfield, RR HIV and beyond.Journal of translational medicine 9, S9 (2011) CCR5 inhibitors in was determined to express the CCR5 receptor at a density [2470±441 molecules/cell (n=6)] consistent with the clinical use of After validating the suitability of the C2C12 system for the assay of the present invention, two human recombinant NAMPT protein sources [hrNAMPT ₍₁₎ and hrNAMPT ₍₂₎ ] were applied to C2C12 myoblasts and proliferation assayed by EdU incorporation. did Both sources of NAMPT produced comparable and significant dose-dependent increases in myoblast proliferation (Fig. 11f). To uncouple the intracellular and extracellular roles of NAMPT during proliferation, these myoblasts were treated with GMX1778, a highly specific and potent inhibitor of NAMPT's enzymatic function. Drug treatment did not adversely affect the basal level of C2C12 proliferation in culture, nor did it affect the increased myoblast proliferation that occurred after exogenous NAMPT supplementation (Fig. 11f). This emphasizes that the growth-promoting role of NAMPT is independent of its intracellular enzymatic function. The enhanced proliferative response observed after NAMPT supplementation could be reproduced in C2C12 cells by the addition of the canonical CCR5 ligands CCL8/MCP-2 and CCL4/MIP-1β, but not the CCR2 ligand CCL2/MCP-1. could not be reproduced in (Fig. 11f). Furthermore, this proliferative response was blocked in the presence of the dual CCR2/CCR5 antagonist senicriviroc (CVC) and the CCR5 selective antagonist maraviroc (MVC), whereas the CCR2 selective antagonist PF-4136309 (PF) was not blocked in the presence of (Fig. 11f). Taken together, this data emphasizes the need for selective signaling of NAMPT through the CCR5 receptor to induce myoblast proliferation. Next, we focused on an in vitro multicellular co-culture system to investigate the relationship between macrophages and NAMPT-mediated proliferation. Isolated embryonic mouse PAX7 ⁺ muscle stem cells were co-cultured with embryonic mouse primary myoblasts alone (MB), mouse myoblasts and mouse macrophages (MB+MΦ), or mouse fibroblasts 3T3 (MB+3T3). was evaluated for proliferation. The murine macrophage cell line utilized is known to express high levels of NAMPT, whereas 3T3 fibroblasts do not naturally secrete NAMPT in culture. In these experiments, the presence of macrophages promoted satellite cell proliferation and no response was elicited when macrophages were replaced in co-cultures by 3T3 cells. Furthermore, addition of hrNAMPT was able to increase satellite cell proliferation under MB and MB+3T3 culture conditions to the same level seen in MB+MΦ cultures, but this effect was abolished in the presence of CVC. Moreover, the addition of CVC reduced the proliferation of satellite cells under MB+MΦ culture conditions to a distinct level in MB alone or MB+3T3 cultures. These results emphasize that increased satellite cell proliferation by macrophages is mediated primarily by secNAMPT acting through the CCR5 receptor. Taken together, these results demonstrate that macrophage-secreted NAMPT is an important proproliferative signal that activates CCR5 receptors present on satellite cells and stimulates muscle regeneration.

These findings were then validated in vivo. The zebrafish ccr5 orthologue was found to be expressed in 2 dpi FACS-sorted pax3a ⁺ myogenic stem/progenitor cells (by RT-PCR) and was used to block Ccr5 signal propagation after pinprick muscle injury. , administered CVC and MVC to zebrafish larvae. Using birefringence imaging, a highly pronounced regeneration defect was confirmed in the drug-treated larvae. This is not due to an effect on macrophage migration velocity or blocking the transition of macrophages from a transient to a resident state, both of which are phenotypically wild-type upon drug administration. there were. Rather, a decrease in proliferation of pax3a ⁺ myogenic stem cells present at the wound site was observed, which was comparable in magnitude to that observed after ablation of resident macrophages. Taken together, these data are consistent with macrophage-secreted nmpt acting in a Ccr5-dependent manner to activate muscle stem cell proliferation during regeneration in vivo.

Mice with an unconditional knockout of the NAMPT gene are embryonic lethal. Therefore, we developed a macrophage-specific loss-of-function mutagenesis system. A Cas9 macrophage-specific stable expression system was prepared by combining the Tg (UAS: NLS-Cas9) transgene and the Tg (mpeg1: Gal4FF) transgene. By delivering nampt guide RNA (gRNA) by microinjection, we achieved durable macrophage-specific nampt gene editing and subsequently assayed the role of macrophage-derived nampt in muscle repair (Fig. 4i, Fig. 8d). . Immunostaining for Nampt revealed that larvae injected with Nampt gRNA had visibly reduced Nampt-expressing cells present at the wound site after needlestick injury. Furthermore, quantification of NAD+/NADH levels in isolated macrophages functionally validated macrophage-specific nampt loss of function using this macrophage-specific gene editing approach.

After needleprick injury, larval macrophages injected with nampt gRNA responded by migrating to the injury site and being located within the wound border between 1-3 dpi. However, these nampt-deficient macrophages fail to induce proper cell proliferation and regeneration at the site of injury, and functional macrophage-derived Nampt is specifically required to ensure proper regeneration. was emphasized. Our in vitro cell culture analysis, together with the results of in vivo chemical inhibition and macrophage-specific nampt loss of function, suggest that macrophage-derived NAMPT is required to stimulate muscle stem cell proliferation in a CCR5-dependent manner. became clear.

Next, the addition of exogenously applied NAMPT stimulates regeneration in mouse models of volumetric muscle loss, an injury paradigm that is normally refractory to normal stem cell-mediated repair processes, and in areas of unexperienced clinical need. [Quarta, M. et al. Bioengineered constructs combined with exercise enhance stem cell-mediated treatment of volumetric muscle loss. Nature Communications 8, 1-17 (2017) (39)]. This analysis confirmed that exogenously applied NAMPT also works in the context of an adult injury model and validated the mammalian cell culture-based results in vivo. Surprisingly, delivery of hrNAMPT in fibrin hydrogels, but not fibrin-only control hydrogels, was able to fully restore muscle architecture when applied to the wound site, which was exogenously supplied. We show that the NAMPT protein stimulates muscle repair in the context of acute muscle injury in adult mammals in a manner similar to the results described above for zebrafish larvae. It also confirms that secreted NAMPT is acting in both of these in vivo settings.

The present invention shows that even this simplest stem cell system requires complex interactions with various cellular contexts during in vivo repair and that the innate immune system, especially macrophages, is a key regulator of the regenerative process. is proposed. In the present invention, there are specific defined macrophage subsets, and the phenotype of the macrophages present during wound repair is surprisingly complex, specifically controlling muscle stem cells through the provision of mitogenic stimuli such as the NAMPT/CCR5 axis. It was shown to act as a transient stem cell niche. Taken together, the present results demonstrate that providing specific macrophage-derived signals required for muscle stem cell proliferation can lead to better therapeutic outcomes with myoblasts and tissue stem cells. .

These results are further supported and illustrated by the following experimental data.

Next, a multicellular in vitro co-culture system was used to examine the relationship between macrophages and NAMPT-mediated proliferation. The proliferation of isolated embryonic mouse PAX7 ⁺ muscle stem cells was expanded with either embryonic mouse primary myoblasts alone (MB), mouse myoblasts and mouse macrophages (MB+MΦ) or mouse fibroblasts 3T3 (MB+3T3). Three conditions of culture were evaluated. Although the murine macrophage cell line utilized (Maf/DKO) is known to express high levels of NAMPT, we aimed to quantify specific levels of NAMPT secretion under the culture conditions of the present invention. rice field. After 16 hours of culture, Maf/DKO macrophages secreted 5.24±0.67 ng/ml of NAMPT into the supernatant (Fig. 11). Moreover, these cells did not actively secrete CCR5's cognate ligands (CCL3, CCL4 and CCL5) into the supernatant (FIGS. 11d-e). On the other hand, 3T3 fibroblasts do not spontaneously secrete NAMPT in culture. In these experiments, we observed that the presence of macrophages promoted satellite cell proliferation (Fig. 4k, Fig. 11g), and this response was not elicited when macrophages were replaced with 3T3 cells in co-cultures (Fig. 4k). , FIG. 11g). Furthermore, addition of hrNAMPT was able to increase satellite cell proliferation under MB and MB+3T3 culture conditions to the same level as that evident in MB+MΦ cultures (Fig. 4k, Fig. 11g), although this effect Similarly, it disappeared in the presence of CVC and was maintained in the presence of PF4 (Fig. 4k, Fig. 11g). These results emphasize that increased satellite cell proliferation by macrophages is mediated primarily by secNAMPT acting through the CCR5 receptor. Interestingly, hrNAMPT did not increase satellite cell proliferation in the presence of macrophages, suggesting that there is a maximum threshold rate of proliferation that may be regulated by receptor saturation. ing. Furthermore, the addition of CCL4 to co-cultures mirrored the results of NAMPT addition, reaffirming that the proliferative effects of NAMPT were mediated by CCR5. These results supported the hypothesis that NAMPT secreted by macrophages activates CCR5 receptors present on satellite cells and is an important growth-promoting signal that stimulates muscle regeneration.

These findings were then validated in vivo. Zebrafish ccr5 orthologues were expressed by RT-PCR at 2 dpi in pax3a+ myogenic stem/progenitor cells isolated by FACS (FIGS. 11h-j), and CVCs and MVCs were expressed in larval zebrafish (4dpf) immediately after needlestick injury. administration to block Ccr5 signaling. Using birefringence imaging, a highly pronounced regeneration defect was detected in drug-treated larvae (FIGS. 11r-s). This is not due to an effect on macrophage or neutrophil migration velocity or a block in the transition of macrophages from the transient to the resident state, all of which become phenotypically wild-type upon drug administration. (Fig. 11k-p). Rather, a decrease in the proliferation of pax3a ⁺ myogenic stem cells residing at the wound site was observed, which was of similar magnitude to that observed after ablation of resident macrophages (Fig. 2f–h, Fig. 11t). u). Furthermore, these results were confirmed by CVC administration, muscle laser ablation, and long-term time-lapse imaging of larvae, which showed no discernible defects in the migration of macrophages and stem cells to the injured area or the transition to a resident state of macrophages. (MΦ onset of normalization at 10.20±0.91 hpi, n=6 injuries). Resident macrophages began binding muscle stem cells by 11.80±0.57 hpi (n=6) and interacted while maintaining continuous uninterrupted binding similar to control macrophages. However, in contrast to untreated larvae, resident macrophages remained associated with their target stem cells until the end of the experiment (18 h, n=6), and these interactions ceased, leading to post-stem cell division. It did not terminate like the control setting where the two cell types separate (Fig. 11q, Supplementary Video 13). Thus, stem cell division may act as a trigger to separate macrophages and their associated stem cells, allowing macrophages to progress to the next target stem cell. Taken together, these data consistently demonstrate that signaling through Ccr5 is required to activate muscle stem cell proliferation during regeneration in vivo.

To provide further evidence for the role of the Nampta/Ccr5 signaling axis in regulating the pro-regenerative, macrophage-derived, muscle stem cell niche, we generated germline mutations in both nampta and Ccr5 genes by CRISPR-Cas9-mediated gene editing. and measured muscle regeneration in homozygous mutants (FIGS. 12a-c, km). Larval zebrafish can survive nampta germline knockout, in contrast to ^mice that are embryonic lethal upon unconditional knockout of the NAMPT gene. This is likely because nmptb is functionally complementary and can at least partially fulfill the protein's enzymatic role in the survival-critical NAD ⁺ salvage pathway ³² . Both mutants showed no comparable difference in numbers of injury-responsive or resident macrophages compared to wild-type or heterozygous siblings (FIGS. 12d-e,no). Moreover, resident macrophages of both mutants became able to interact with muscle stem cells located at the wound site (Fig. 12f,p). Both nampta and ccr5 homozygous mutants scored markedly defective regeneration using birefringence analysis after pinprick injury (Fig. 12g-h, qr). Moreover, in both cases, this defect was due to a marked reduction in proliferating muscle stem cells within the injured area (FIGS. 12i-j, st).

To clarify whether macrophage-derived Nampta is required for regeneration-promoting activity, we developed a novel macrophage-specific loss-of-function mutagenesis system (AII, Z. Zhang, V. Pazhakh, HR Manley, ER Thompson, LC Fox, S. Yerneni, P. Blombery, GJL, in writing). Stable expression of macrophage-specific Cas9 protein was generated from a combination of Tg(4xUAS:NLS-Cas9) and Tg(mpeg1:Gal4FF) transgenes. Delivery of nampta guide RNA (gRNA) by microinjection revealed durable macrophage-specific nampta gene editing with sequence disruptions at and around nampta gRNA target sites in gene-edited macrophages ( Figure 13a). Immunostaining for nampta also confirmed a visible reduction in Nampt-expressing cells present at the wound site after needlestick injury in nampta-gRNA-injected larvae (Fig. 13b). Furthermore, quantification of NAD ⁺ /NADH levels in isolated macrophages functionally validated macrophage-specific nampta loss of function using this macrophage-specific gene editing approach (Fig. 13c).

Using this validated lineage-specific gene-editing method, the role of macrophage-derived Nampta in muscle repair was assessed using our standardized muscle injury paradigm (Fig. 4b). After pinprick muscle injury, larval macrophages injected with nampta-gRNA responded by migrating to the injury site, transitioning to the resident subtype, and actively interacting with muscle stem cells at the injury site (Fig. 13d-g). ). However, these nampta-deficient macrophages failed to induce adequate cell proliferation and regeneration at the site of injury (Fig. 4d–g), and functional nampta-derived macrophages were required to ensure adequate regeneration. It was highlighted that it is particularly necessary. Additional control experiments were performed for the mpeg-Cas9 experiment and a new experiment inhibiting NAMPT enzymatic activity to demonstrate that it did not adversely affect muscle stem cell proliferation, in contrast macrophage-specific namptb knockdown larvae It undergoes significant muscle regeneration compared to controls, albeit at a slower rate than that evident in control injured larvae (Extended Data Figures 13l-n). Moreover, neutrophil-specific deletion of nampta using the Tg(mpx1:KALTA4);Tg(4xUAS:NLS-Cas9) transgene combination did not result in any defects in muscle regeneration (Fig. 13o-q ), reconfirming that macrophages are the source of Nampta present at the wound site. Finally, we investigated whether Nampt's pro-regenerative cytokine activity was independent of its enzymatic function in vivo, as revealed in in vitro-based assays. Addition of GMX1778 to regenerating larvae and inhibition of the intracellular enzymatic function of Nampt by strict temporal control from the time macrophages began to reside did not change the proliferation of muscle stem cells (Fig. 10p-q). This supported the in vitro finding that the secretory form of Nampt dominates its proliferative function. Taken together, our in vitro cell culture analysis and the results of studies of in vivo chemical inhibition and macrophage-specific nampta loss of function suggest that macrophage-derived NAMPT is required to stimulate muscle stem cell proliferation in a CCR5-dependent manner. was revealed (Fig. 14j).

Experiments were then designed to confirm that the Ccr5 receptor is specifically required for muscle stem cells to stimulate their proliferation during the regeneration process. To perform these analyses, we used the pax7b:GAL4FF strain described above, which is expressed in muscle stem cells present at the wound site (Fig. 7c). Expression of the 4xUAS:NLS-Cas9 transgenic line with this pax7b promoter enabled muscle stem cell-specific ccr5 knockdown using highly efficient dual gRNA combinations. This muscle stem cell lineage-specific ablation ccr5 knockdown did not alter the numbers of transient and resident macrophages at the injury site, nor did it affect the association of resident macrophages with stem cells (Fig. 13h). ~j). These gene-edited larvae exhibited marked defects in regeneration after needlestick muscle injury (Fig. 4c-e) and marked defects in muscle stem cell proliferation at the wound site (Fig. 4h-i). These defects were of similar magnitude to defects revealed in germline ccr5 mutants (Fig. 12k-t) and larvae in which Ccr5 activity was chemically inhibited (Fig. 11r-s). Taken together, these data demonstrate that muscle stem cell Ccr5 activity is required to induce stem cell proliferation during muscle regeneration.

Next, the effect of adding exogenous-NAMPT to larval zebrafish after needlestick injury was examined. Since previous studies have shown that secretory NAMPT may inhibit neutrophil apoptosis ⁵¹ , thereby altering inflammatory dynamics, we analyzed the response of innate immune cells to NAMPT supplementation. In our acute injury setting, we did not observe changes in immune cell dynamics or lysosomal activity at the wound site with exogenous NAMPT treatment (FIGS. 14a-c). However, NAMPT supplementation resulted in a highly significant increase of 30.70±4.26% in muscle stem cell proliferation within the wound site (Fig. 4j, Fig. 14d). Furthermore, NAMPT supplementation also functioned to rescue wound site proliferation of macrophage ablation larvae, even acting to increase proliferation by 10.96±3.55% over responses in control settings (Fig. 4j, Figure 14d). However, NAMPT was unable to rescue the proliferation defect when co-replenished with CVC (Fig. 4j, Fig. 14d). Interestingly, CCL8, the canonical Ccr5 ligand, was able to enhance the wound site proliferative response to the same extent as NAMPT (on average 28.34±4.48% higher than control), but compared with control. also increased cell proliferation outside the wound site by 43.03±4.46% (Fig. 4j, Fig. 14d). This emphasizes that NAMPT has a specific function on muscle stem cells present at the wound site, distinct from the general proliferative response elicited by Ccr5 activation.

Next, we examined whether the addition of exogenous NAMPT promotes regeneration in volumetric muscle weakness model mice, an injury paradigm that is difficult to repair by endogenous stem cells and is an unsolved clinical problem ⁵² . This analysis confirmed that exogenous NAMPT also works in an adult injury model, and validated mouse cell culture-based results in vivo. Surprisingly, delivery of hrNAMPT to the muscle defect via a fibrin hydrogel, but not a fibrin-only control hydrogel, and application to the wound site was able to fully restore muscle architecture (Fig. 4l-o). ). On average, treatment with a single dose of hrNAMPT (0.5 μg) at the time of injury increased mean muscle area by 3.276±0.4926 ^mm2 and mean fibrotic area by 34.76±9.32%. Diminished. NAMPT addition in the VML injury model resulted in a significant increase in both the total number and percentage of proliferating PAX7 ⁺ satellite cells (Fig. 4p-r, Fig. 14e), with a significant increase in the number of de novo myofibers with central nuclei. (Fig. 4s-t) but did not induce major changes in the immune cell profile of the regenerates (Fig. 14h-i). Although neovascularization within the wound occurred at levels similar to those described for other pro-regenerative ^approaches in VML injury52,53 (Extended Data Fig. 14f-g), this suggests that neovascularization was only due to NAMPT treatment. suggest that it is proportional to the level of regeneration apparent in the injured lesion. However, the more direct mode of action of NAMPT and CCR5 to stimulate angiogenesis, especially given that these proteins have previously been shown to induce endothelial cell proliferation in many different contexts, is It cannot be formally ^denied54-56 . Taken together, these findings suggest that exogenously supplied NAMPT protein can stimulate muscle repair in the context of acute adult mammalian muscle injury, similar to the results described above for zebrafish larvae. shown. It also reinforced the finding that it is the secreted form of NAMPT that is active in these in vivo settings.

NAMPT Fragments and Derivatives Thereof In one embodiment, dimers are formed by adding a cysteine residue at the N-terminus (with or without a linker, eg, GGS or GGS repeats). A cysteine can also occur naturally in a NAMPT sequence.
CSYVVTNGLGINVFKDPVADPNKRSKKGRLSLHRTPAGNFVTLEEGKGDLEEYGQDLLHTVFKNGKVTKSYSFDEIRKNAQLNIELEAAHH
Human:
SYVVTNGLGINVFKDPVADPNKRSKKGRLSLHRTPAGNFVTLEEGKGDLEEYGQDLLHTVFKNGKVTKSYSFDEIRKNAQLNIELEAAHH
mouse:
SYVVTNGLGVNVFKDPVADPNKRSKKGRLSLHRTPAGNFVTLEEGKGDLEEYGHDLLHTVFKNGKVTKSYSFDEVRKNAQLNIEQDVAPH

The C-terminal fragment of NAMPT stimulates muscle stem cell proliferation. NAMPT is an intracellular enzyme having a large homodimer as described above, and acts as a cytokine when released outside the cell. However, the NAMPT domain responsible for cytokine activity remained unknown. A review of the crystal structure of NAMPT revealed that the structure of the C-terminus of NAMPT is very similar to classical CCR-binding chemokines (such as CCL2) due to its size and structure (C-terminal α-helix and β-sheet). (Fig. 9a). Furthermore, this domain extends from the core protein structure and may facilitate binding to receptors. Therefore, the C-terminus of NAMPT was recombined and tested for its ability to compete with NAMPT binding to CCR5 and stimulate satellite cell proliferation. Surprisingly, this fragment, referred to herein as the "cytokine finger" (cif), inhibited NAMPT binding to CCR5 (IC50 = 21.5 nM, Fig. 9b) and dose-dependently increased satellite cell proliferation. and induced growth at the same level as full-length NAMPT (Fig. 9c). Taken together, these data indicate that the C-terminal cif domain is involved in the muscle cytokine activity of NAMPT.

A NAMPT cytokine derivative aimed at clinical application. NAMPT derivatives are engineered with various modifications to improve delivery and enhance receptor-mediated activity. These forms are serially assayed for clinically useful properties such as increased or optimized receptor binding affinity, in vitro cell-based proliferation, and in vivo zebrafish or mammalian muscle regeneration assays. . Tissue targeting is clearly of great value in multiple modes of administration, and the inclusion of tissue-directing moieties is proposed to enhance delivery and proper retention in target tissues. NAMPT or NAMPT derivatives can also be administered by carriers such as nanocarriers known in the art to provide tissue targeting through their composition or functionalized structure.

Dimerizes NAMPT cytokine fingers and increases receptor affinity. NAMPT is a homodimer in nature, the known CCR5-binding chemokines are dimers, and they form multimers (trimers, tetramers, oligomerization and higher) that regulate receptor binding affinity and signal transduction. ) can be formed. In one embodiment, NAMPTcif is dimerized to increase its binding affinity to CCR5 (Fig. 9d). In one approach, a single naturally occurring cysteine residue at the N-terminus of NAMPTcif is used to force its dimerization. The binding affinity of dimeric NAMPTcif for CCR5 is tested by ELISA and surface plasmon resonance (SPR) assays. The activity of dimeric NAMPTcif to promote proliferation of mouse primary satellite cells was tested. Quiescent satellite cells have negative and positive cell surface markers (CD31-, CD11b-, CD45-, TER119-, Sca1-, CD34+) specific for a subset of satellite cells shown to have the highest self-renewal properties in vivo. , CD106+) and are sorted from fresh muscle as described above. Cells are cultured in vitro to determine the effect of NAMPT derivatives on stimulating proliferation of these primary muscle stem cells. The regeneration ability of dimeric and multimeric NAMPTcif, and functional derivatives, is tested in the described zebrafish regeneration assay.

Addition of ECM- and/or syndecan-binding motifs is used as one of the preferred approaches to optimize delivery and increase tonic signaling on CCR5 (see Mochizuki et al Nat. Biomed Engineering 2019) (Fig. 14). Some proteins bind to syndecans such as laminin. One particular syndecan binding site is the globular domain of the laminin-α chain with the sequence RKRLQVQLSIRT (SB). Addition of binding molecules may be by synthetic means or using recombinant approaches, as is known in the art. Exemplary, non-limiting ECM binding sites include RGD, or YGISR, YIGSR, GFOGER, IKVAV, and GEFYFDLRLKGK.

One of the models suitable for testing functional derivatives is the established model of cardiomyotoxin-induced muscle damage. Cardiotoxins are myonecrolytic agents that kill myocytes without destroying the muscle ECM, providing an important model for testing NAMPT variants containing ECM-binding motifs. It is the most commonly used model when assaying muscle stem cell activation because it leaves the majority of stem cells intact. A standard model is the intramuscular injection of cardiotoxin into the tibialis anterior muscle (TA) and assay of recovery of lost fibers. Finally, ARMI-established mdx mice will be tested. The mdx model is used to test both the stem cell activating and anti-fibrotic potential of NAMPT derivatives, as it shows chronic muscle fibrosis concurrently with muscle degeneration. Muscle regeneration is tested at established time points using the standard histological assays described above.

Satellite cells are prototypical in that they are unipotent, tissue-resident stem cells that occupy specific anatomical niches within differentiated tissues. Recent decades of research have revealed the unique ability of this system to effectively coordinate muscle repair in response to a wide variety of injuries. Despite this demonstrated regenerative capacity, transplantation of isolated muscle stem cells has not yet had therapeutic impact, and regenerative therapies that stimulate muscle stem cells are still lacking. The data disclosed in the present invention demonstrate that even this simplest stem cell system requires a complex interaction with various cellular contexts during in vivo repair, and that the innate immune system, especially macrophages, is critical in the regenerative process. proposed to be a regulatory factor. What we have shown here is that a specific macrophage subset, one of the surprisingly complex macrophage phenotypes present during wound repair, activates muscle stem cells through the supply of mitogenic stimuli, particularly the NAMPT/CCR5 axis. functioning as a transient stem cell niche that directly regulates (Fig. 14j). Thus, providing specific macrophage-derived signals required for muscle stem cell proliferation, such as the signals identified in the present invention, provide an avenue for improving myoblast-based therapeutic outcomes. be.

＊＊注：コドンは置換可能であり、コドンの置換については置換表を参照されたい。

**Note: codons are permutable, see substitution table for codon substitutions.

In some embodiments, TRP residues are substituted.

to all documents cited or referenced herein, and to all cited documents cited or referenced herein, and to any product described herein or in a document incorporated by reference herein; The manufacturer's instructions, descriptions, product specifications and product sheets are hereby incorporated by reference in their entireties. The entire contents of Australian Provisional Patent Application No. 2020904717 filed on December 17, 2020 and Australian Provisional Patent Application No. 2020901237 filed on April 20, 2020 are hereby incorporated by reference in their entireties. be The drawing is also clearly represented in color in the paper published by the inventors in Nature 2021 Mar;591 (7849): 281-287. doi: 10.1038/s41586-021-03199-7. Epub 2021 Feb 10.

Those skilled in the art will appreciate, in light of the disclosure herein, that various modifications and changes can be made to the specific embodiments illustrated without departing from the scope of the invention. All such modifications and changes are intended to be included within the scope of the appended claims.

References
Gurevich, DB et al. Asymmetric division of clonal muscle stem cells co-ordinate muscle regeneration in vivo. Science (2016).

Novak, ML & Koh, TJ Macrophage phenotypes during tissue repair. Journal of leukocyte biology 93, 875-881 (2013).

Yahiaoui, L., Gvozdic, D., Danialou, G., Mack, M. & Petrof, BJ CC family chemokines directly regulate myoblast responses to skeletal muscle injury. The Journal of physiology 586, 3991-4004 (2008).

Grolla, AA, Travelli, C., Genazzani, AA & Sethi, JK Extracellular nicotinamide phosphoribosyltransferase, a new cancer metabokine. British journal of pharmacology 173, 2182-2194 (2016).

Van den Bergh, R. et al. Monocytes contribute to differential immune pressure on R5 versus X4 HIV through the adipocytokine visfatin/NAMPT. PloS one 7, e35074 (2012).

Quarta, M. et al. Bioengineered constructs combined with exercise enhance stem cell-mediated treatment of volumetric muscle loss. Nature communications 8, 1-17 (2017).

Mochizuki et al., Nat. Biomed Engineering 2019.

Claims (49)

A method of stimulating muscle tissue regeneration comprising administering to muscle an effective amount of a composition comprising or encoding a CCR5 interacting agent and, optionally, a component that enhances delivery to muscle, wherein , a CCR5-interacting agent binds to muscle stem cells and stimulates myoblast proliferation and muscle regeneration. 2. The method of claim 1, wherein the CCR5 interacting agent is a CCR5 agonist. 3. The method of claim 2, wherein the CCR5 agonist activates the Mapk/Erk signaling cascade. 4. The method of claim 1, 2 or 3, wherein the CCR5 interacting agent specifically activates stem cells. 5. The method of any one of claims 1-4, wherein muscle regeneration is associated with no or minimal fibrosis. 6. The method of any one of claims 1-5, wherein the muscle tissue is in vitro, in vivo or ex vivo. 7. The method of any one of claims 1-6, wherein the CCR5 interacting agent competes with nicotinamide phosphoribosyltransferase (NAMPT) for interaction with muscle stem cells. 8. A method according to any one of claims 1 to 7, wherein the CCR5 interacting agent is NAMPT or a portion or derivative thereof comprising a cytokine finger (cif) motif. 9. The method of any one of claims 1-8, wherein the composition is a cell composition comprising cells expressing a CCR5 interacting agent. 10. The method of claim 9, wherein the cells are macrophages. 11. The method of claim 9 or 10, wherein the composition further comprises stem cells. 12. A method according to any one of claims 1 to 11, wherein the CCR5 interacting agent in the composition is NAMPT or a portion or derivative thereof comprising a cytokine finger (cif) motif. 13. The method of claim 12, wherein the NAMPT or part or derivative thereof comprises one or more moieties such as linkers, stability or signaling enhancing, delivery enhancing or labeling moieties. 13. The method of claim 11 or 12, wherein NAMPT or a portion or derivative is in monomeric, dimeric or multimeric form. 2. The method of claim 1, wherein the composition encoding the CCR5 interacting agent comprises a nucleic acid molecule that expresses the CCR5 interacting agent. 16. The method of claim 15, wherein the nucleic acid molecule is RNA or DNA or RNA:DNA or chemically modified forms thereof. 17. A method according to claim 15 or 16, wherein the nucleic acid is in the form of a viral or non-viral vector. 9. The method of any one of claims 1-8, wherein the CCR5 interacting agent is a small molecule. 9. The method of any one of claims 1-8, wherein the CCR5 interacting agent is an antibody or comprises a CCR5 binding portion thereof. A composition comprising or encoding a CCR5 interacting agent as defined herein or according to any one of claims 1-19. A pharmaceutical or physiologically active regenerating composition comprising 1 or 2 or 3 or 4 or 5 of:
(i) comprises or encodes a CCR5 interacting agent as defined herein;
(ii) satellite cells or their progenitor cells or their progeny cells (iii) macrophages or their progenitor cells or their progeny cells (iv) scaffolds or other retaining materials (v) tissue delivery enhancing components. 20. A CCR5 interacting agent as defined herein or according to any one of claims 1 to 19, comprising or encoding for use in stimulating muscle regeneration in vitro, ex vivo or in vitro A composition that has become 23. The composition of claim 22, wherein the CCR5 interacting agent is NAMPT or its C-terminal portion comprising a cytokine finger (cif) motif, or a functional derivative thereof. 23. A composition according to claim 22, for use in artificial muscle production (e.g. meat, including fish or poultry, for direct consumption or further processing). 23. The composition of claim 22 for use in stem cell therapy or for the manufacture and administration of stem cell-based therapeutics. 22. For use in the treatment of a muscular, neuromuscular or musculoskeletal defect, disorder or injury, or for use in the treatment of muscle tissue regeneration associated with fibrosis to reduce fibrosis. The composition according to . A NAMPT polypeptide fragment comprising the C-terminal portion of NAMPT containing a cytokine finger (cif) motif, or a modified form thereof, or a functional derivative thereof. 28. A NAMPT polypeptide fragment or modified form thereof according to claim 27, comprising a peptide sequence set forth in SEQ ID NO: 1 or 2 or 5, or a sequence having at least 80% identity to SEQ ID NO: 1 or 2 or 5 Or a functional derivative.
[Claim 28]
29. The NAMPT polypeptide fragment of claim 27 or 28, comprising one or more of a linker, a stability-enhancing moiety, a signaling-enhancing moiety, a delivery-enhancing moiety, a tissue or muscle retention-enhancing moiety, and a labeling moiety. 29. A NAMPT fragment according to claim 27 or 28, in monomeric, dimeric or multimeric form. A composition comprising a NAMPT polypeptide fragment or derivative according to any one of claims 27 to 30, or a nucleic acid molecule encoding them, and one or more of the following:
(i) tissue stem cells or their progenitors or their progeny (ii) macrophages or their progenitors or their progeny (iii) scaffolds (semi-solid or solid supports) or retention materials (iv) tissue delivery enhancing or cell retention moieties . 31. The composition of claim 30, wherein the scaffolding or holding material is fibrin or a hydrogel such as an acrylamide hydrogel.
[Claim 31]
32. The composition of claim 30 or 31 or the NAMPT functional derivative of any one of claims 27-29, wherein the tissue delivery enhancing moiety or cell retention moiety is an ECM binding moiety. 32. A composition according to claim 30 or 31 or a NAMPT functional derivative according to any one of claims 27 to 29, wherein the signaling enhancing moiety is a heparin sulfate binding moiety, such as a syndecan binding moiety. A method of stimulating tissue regeneration, wherein an isolated or tissue-resident tissue stem cell or its progenitor cell comprises or encodes a CCR5-interacting agent, and optionally a component that enhances delivery to the tissue. A method comprising administering an effective amount of a composition, wherein the CCR5 interacting agent binds to tissue stem cells and stimulates (activates) quiescent tissue stem cell proliferation and tissue regeneration. 34. The method of claim 33, wherein the CCR5 interacting agent is a CCR5 agonist. 35. The method of claim 34, wherein the CCR5 agonist activates the Mapk/Erk signaling cascade. 36. The method of any one of claims 33-35, wherein the CCR5 interacting agent competes with nicotinamide phosphoribosyltransferase (NAMPT) for interaction with muscle stem cells. 37. A method according to any one of claims 33 to 36, wherein the CCR5 interacting agent is NAMPT or a portion or derivative thereof comprising a cytokine finger (cif) motif. 38. The method of any one of claims 33-37, wherein the composition is a cell composition comprising cells expressing a CCR5 interacting agent. 39. The method of claim 38, wherein the cells are macrophages. 40. The method of claim 38 or 39, wherein the composition further comprises stem cells. 41. A method according to any one of claims 33 to 40, wherein the CCR5 interacting agent of the composition is NAMPT or a portion or derivative thereof comprising a cytokine finger (cif) motif. 42. The method of claim 41, wherein NAMPT or a portion or derivative thereof comprises one or more moieties such as linkers, stability or signaling enhancing, delivery enhancing or labeling moieties. 43. The method of claim 41 or 42, wherein NAMPT or a portion or derivative is in monomeric, dimeric or multimeric form. 34. The method of claim 33, wherein the composition encoding the CCR5 interacting agent comprises a nucleic acid molecule that expresses the CCR5 interacting agent. 45. The method of claim 44, wherein the nucleic acid molecule is RNA or DNA or RNA:DNA or chemically modified forms thereof. 46. The method of claim 45, wherein the nucleic acid is in the form of a viral or non-viral vector. 37. The method of any one of claims 33-36, wherein the CCR5 interacting agent is a small molecule. 37. The method of any one of claims 33-36, wherein the CCR5 interacting agent is an antibody or comprises a CCR5 binding portion thereof. 49. The method of any one of claims 33-48, wherein tissue regeneration occurs without or substantially without fibrosis.

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